Methods for treatment for ulcerative colitis in mammals

ABSTRACT

The subject invention pertains to materials and methods for the prevention and treatment of disease conditions associated with oxidative stress or a compromised reducing environment, including inflammatory bowel diseases such as ulcerative colitis. Another aspect of the subject invention concerns compositions formulated for administration as an enema. The subject invention also concerns compositions formulated for oral administration. Methods of the invention include administration of compounds or compositions of the invention. In one embodiment, compounds or compositions of the invention are rectally instilled in a patient. In another embodiment, compounds or compositions are orally administered.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the National Stage of InternationalApplication No. of PCT/US2008/007401, filed Jun. 13, 2008, which claimsthe benefit of U.S. Provisional Patent Application Ser. Nos. 60/934,505,filed Jun. 13, 2007 and 61/063,745, filed Feb. 6, 2008. The presentapplication is also a continuation-in-part of U.S. application Ser. No.11/540,864, filed Sep. 28, 2006, which is a continuation-in-part of U.S.application Ser. No. 11/107,179, filed Apr. 15, 2005, which is acontinuation-in-part of U.S. application Ser. No. 10/927,742, filed Aug.27, 2004, now U.S. Pat. No. 7,312,243, which claims the benefit of U.S.Provisional Application Ser. No. 60/499,152, filed Aug. 29, 2003, eachof which is hereby incorporated by reference herein in its entirety,including any figures, tables, nucleic acid sequences, amino acidsequences, and drawings.

BACKGROUND OF THE INVENTION

Ulcerative colitis is an inflammatory bowel disease characterized byrecurrent bouts of rectal bleeding and bloody diarrhea. The initialinflammatory reaction begins in the rectal mucosa in over 95% of casesand may extend in a contiguous fashion to involve the whole colon(Hendrickson, 2002).

Histologically, ulcerative colitis is manifest by mainly neutrophilinfiltration into the colonic mucosal crypts of Lieberkuhn leading to aneutrophilic cryptitis and the formation of micro crypt abscesses, whichcoalesce to form bleeding macroscopic mucosal ulcerations. Neutrophilicsecretion of tissue destructive cytokines and oxygen radicals leads to achronic crypt destructive colitis that can involve the entire colon(Carpenter, 2000).

Treatment modalities are few and unsatisfactory and remain confined toaminosalicylate derivatives and anti-inflammatory corticosteroids forinitial therapy, progressing to potent immunosuppressive agents forrecalcitrant disease and finally to colectomy for those patientsunresponsive to medical therapy. Most patients with mild to moderatedisease have an unpredictable course. Individuals with severe diseasecomprise approximately 20% of patients. About 85% of patients withsevere or fulminant disease will undergo total colectomy within a year.The cumulative likelihood of requiring colectomy by 25 years is about32%. (National Guidelines Clearinghouse: http://www.ngc.gov “Managementof Ulcerative Colitis”).

Medical treatment strategies for ulcerative colitis have been directedtowards either neutralizing one or more of the cytokines produced by theinfiltrating neutrophils or eliminating the source of the cytokine,i.e., the neutrophil itself. Since the history of medically treatedulcerative colitis is characterized by lifelong repeated episodes of thedisease, it appears that no currently available medical therapeuticmodality is capable of addressing the fundamental disorder present andtherefore current therapies are unable to alter the natural history ofthis condition.

It is perhaps among the greatest physiological wonders of evolution thatthe most highly evolved immune system ever engendered can remainunperturbed while surrounding the highest concentration of bacteria onthe planet, separated only by a tenuous sheet of tissue one cell thick.

This unlikely truce describes the living conditions of the normal humancolon where the luminal concentration of potentially pathogenic bacteriais estimated to be 10¹² (one trillion) colony-forming units (viablebacterial cells) per gram of colonic contents (Farrell and Peppercorn,2002). The number of prokaryotic bacterial cells in the gastrointestinaltract (10¹⁴) (one hundred trillion) is equal to the total number ofcells in the human body (Blaut, 2000; Guyton and Hall, 1997). Close toone half of the weight of feces produced is composed of bacteria andthere are over 400 known species of bacteria in the normal human colon,many of which are quite virulent and pyogenic if translocated to otherbody cavities outside of the intestine.

The mucosal immune system of the gastrointestinal tract can beconceptually divided into a normally non-reactive or tolerant surfacecomponent and a potentially reactive sub-surface entity. The surfacecomponent, consisting of T-cells interspersed among the colonicepithelial cells, has been rendered tolerant to colonic bacteria. Thisanergic surface T cell response to normal luminal bacterial flora hasbeen present since birth. Teleologically, these T-cells may serve as afirst line defense to recognize foreign bacteria that infect the mucosaitself.

The reactive component of the sub-surface immune system consists ofmacrophages, B-cells and additional T-cells that reside within thenormally sterile environment just beneath the colonic epithelialbasement membrane in the lamina propria. These immune cells arephysically separated from luminal bacterial antigens by three distinctphysical barriers of protection. These consist of, starting from theluminal side, a protective mucus layer, an intact colonic mucosal liningand subjacent basement membrane. This degree of separation maintains asterile sub-epithelial environment and shields the lamina propria immunecells and vasculature from encountering colonic bacterial products thatwould otherwise initiate an immune and chemotactic response.

The integrity of the colonic epithelial cell/basement membrane (surface)barrier is paramount in maintaining immune quiescence within the colonictissues and preventing the colonic immune system from mounting an immuneresponse to the high concentration of bacterial antigen that is poisedto invade the normally sterile sub-epithelial environment.

Cellular mechanisms involved in maintaining the integrity of the colonicsurface barrier function may therefore be compromised early on in thepathogenesis of ulcerative colitis. Dysfunction of a vital processrequired to maintain mucosal integrity must therefore be an early andnecessary part of a sequential series of events ultimately leading todeterioration of epithelial barrier function with subsequent mucosalimmune activation secondary to antigenic penetration into the laminapropria.

In other words, the additive effect of abnormal cellular stressorsfocused on a common biochemical pathway are acting in concert to disruptan intracellular biochemical process that contributes a requiredfunction necessary for maintaining colonic surface barrier integrity.

The high incidence (over 50%) of spontaneous improvement and relapseseen in ulcerative colitis (Meyers and Janowitz, 1989) suggests areversible disruption and the possibility of a self replenishingdepletion syndrome affecting a crucial element required for mucosalintegrity.

Experimental attempts to create an animal model of human ulcerativecolitis using rectal instillation of toxic chemicals are inherentlylimited in their ability to faithfully reproduce the disease due tocomplex psychological, physiological, genetic, environmental andimmunological interactions that antecede and contribute to thepathogenesis of this condition in humans (Farrell and Peppercorn, 2002).In vivo human colonic exposure to toxic chemicals is not currentlyadvocated for any clinical condition. However, such was not always thecase.

For many years during the twentieth century hydrogen peroxide enemaswere routinely employed and recommended by physicians for the evacuationof fecal impactions. As recently as 1980, hydrogen peroxide enemas werebeing advocated for the treatment of fecal impaction in a major nursingtext (Brunner and Suddarth, 1980). However, in the 1930's reports beganto surface regarding the development of rectal bleeding and colitissubsequent to the use of hydrogen peroxide enemas (Benson and Bargen,1939). A fatal case of colitis subsequent to hydrogen peroxide enema wasfirst recorded in 1948 (Sheenan and Brynjolfsson, 1960). In this case,the authors report a 41-year-old white female who died 5 days afterself-administration of a hydrogen peroxide enema to relieve a fecalimpaction. The autopsy report noted acute ulcerative colitis, which was“attributed to the action of the hydrogen peroxide enema.” Since thenthere have been reports of fatal outcomes secondary to the developmentof colitis subsequent to the use of hydrogen peroxide enemas. In 1951,Pumphrey reports severe ulcerative proctosigmoiditis following hydrogenperoxide enemas in two patients (Pumphery, 1951). In 1967, Shaw reportedthe deaths of several infants some time following the evacuation ofimpacted meconium with hydrogen peroxide (Shaw et al., 1967). In 1981,Meyer et al. reported three cases of acute ulcerative colitis afteradministration of hydrogen peroxide enema and stated that “acuteulcerative colitis appears to be a fairly predictable occurrence afterhydrogen peroxide enemas” (Meyer et al., 1981). In 1989, Bilotta andWaye describe an epidemic of hydrogen peroxide induced colitis in theG.I. endoscopy unit at their institution. This was due to theinadvertent instillation of hydrogen peroxide during colonoscopy. Uponcontact of the hydrogen peroxide with colonic mucosa they visualizedinstantaneous mucosal whitening and frothy bubbles, which they describeas the “snow white” sign (Bilotta and Waye, 1989). In 1995, inadvertentcolonic instillation of hydrogen peroxide during colonoscopy resulted inthe same mucosal reaction (Schwartz et al., 1995). In 2001, rectalbleeding was, once again, noted to be a complication of hydrogenperoxide enemas (Thibaud et al., 2001).

In their classic experiments, Sheehan and Byrnjolfsson (1960) producedacute and chronic ulcerative colitis by rectal injection of rats with a3% solution of hydrogen peroxide. Microscopic examination of sacrificedrats revealed colonic mucosal ulceration and neutrophilic infiltration,which was “sharply delineated from adjacent normal mucosa.” The mucosalinflammation, which reached 5 cm above the anus at 5 hours afterinjection had extended proximally to 9 cm by one week. Additionally theauthors noted that, in surviving rats, most of the colonic mucosalulcerations were healed by 10 weeks with the exception of some, which“were located almost always in the left colon a few centimeters abovethe anus.”

Hydrogen peroxide is a colorless, heavy, strongly oxidizing liquid, apowerful bleaching agent; also used for wastewater treatment, as adisinfectant and as an oxidant in rocket fuels. Hydrogen peroxide (H₂O₂)also has a ubiquitous presence in cells and is continuously beinggenerated in the cytosol and several different sub-cellular organellesincluding peroxisomes, endoplasmic reticulum and nucleus by variousoxidase and oxygenase enzymes (i.e. xanthine oxidase, cytochrome p450oxygenase) (Chance et al., 1979). However, in most cells, approximately90% of hydrogen peroxide is generated as a toxic by-product ofmitochondrial electron transport chain respiratory activity (Eaton andQian, 2002).

The mitochondrial electron transport chain (ETC) consists of fivedistinct protein components, which are embedded within the mitochondrialinner membrane facing the inner liquid matrix. Three of these componentsare large, membrane fixed, protein complexes (Complex I, III and IV),which serve as trans-membrane redox linked proton pumps that act totransfer protons from the matrix through the inner membrane into theinter-membrane space (Schultz and Chan, 2001). These three complexesinteract with two smaller mobile carriers (complex II and cytochrome c),which shuttle electrons between the complexes. Complex II (Succinatedehydrogenase, EC 1.3.5.1) transfers electrons between Complex I (NADHdehydrogenase EC 1.6.5.3) and complex III (Ubiquinol-cytochrome creductase, EC 1.10.2.2) while cytochrome c (a small heme containingprotein) shuttles electrons from complex. III to complex IV(Cytochrome-c oxidase, EC 1.9.3.1). These redox electron transfersresult in conformational changes of the inner membrane-bound proteincomplexes (I, II and III), which drive the flow of protons from thematrix through the inner membrane and into the inter membrane space. Theresultant accumulation of protons within the inter membrane spacecreates an electrochemical gradient, which drives the flow of theseprotons back into the matrix through a trans-membrane enzyme (ATPsynthase, EC 3.6.1.34 or Complex V). It is the energy provided by thisretrograde flow of protons down its electrochemical gradient, whichprovides the energy for ATP synthase to synthesize ATP (Schultz andChan, 2001). The final acceptor of electrons in the chain is diatomic(molecular) oxygen, which is completely reduced to water by Cytochrome-coxidase (complex IV) in a reaction in which molecular oxygen (O₂)combines with 4 electrons (e⁻) and 4 protons (H+) to produce twomolecules of water (H₂O). Complex I and III are the source of electronleakage leading to the eventual intracellular generation of hydrogenperoxide (Lemasters and Nieminen, 2001; St.-Pierre et. al., 2002).

There are thousands of these electron transport chain protein complexesdoting the matrix aspect of mitochondrial cristae, which continuouslyreduce oxygen in order to build the electrochemical potential needed tocreate a chemiosmotic gradient of protons in the intermembrane spacethat drives the synthesis of adenosine triphosphate (ATP).

The transfer of electrons through the electron transport chain, however,is not perfect and up to 5% of electrons do not make it all the waythrough the chain and fail to combine with oxygen to produce water (Liu,1997; Turrens, 1997; Eberhardt, 2001). Electron transfer through the ETCdepends upon requisite conformational changes and proton transfers whichmust occur prior to the electron passing to the next protein in thechain. Failure of these changes to take place leads to a decoupling ofelectron transfer referred to as an electron leak (Schultz and Chan,2001). These “leaked” electrons, from complex I and III of the electrontransport chain, combine directly with molecular oxygen in the immediatevicinity, instead of the next carrier in the chain, to form superoxide(O₂ ⁻.) which is the first (single electron) incomplete reductionproduct of molecular oxygen (Cadenas and Davies, 2000). The extraunpaired electron in its outer valence orbital makes superoxide aradical, also commonly referred to as a reactive oxygen metabolite (ROM)or species (ROS). It is estimated that 2% of available oxygen isconverted to superoxide by electron transport chain “leakage” (Boverisand Chance, 1973). At physiological pH superoxide exists as an anionradical and acts preferentially as a reducing agent (donate an electron)(Eberhardt, 2001). Superoxide can cause serious damage to cells ifallowed to accumulate.

Superoxide, however, due to its negative charge, cannot pass throughbiological membranes and is contained within the mitochondria.Superoxide can spontaneously dismutate to hydrogen peroxide or undergoenzymatic dismutation to hydrogen peroxide (H₂O₂) at the site ofproduction within mitochondria by the enzyme superoxide dismutase (SOD)(EC 1.15.1.1) (Chance 1979, Eberhardt, 2001). In this enzymatic reactiontwo superoxide molecules are combined with two protons and converted toone molecule of hydrogen peroxide and one molecule of diatomic oxygen,(O₂ ⁻.+O₂ ⁻.→SOD, 2H⁺→H₂O₂+O₂). Superoxide is considered to be astoichiometric precursor of mitochondrial hydrogen peroxide (Chance etal., 1979; Han et al., 2001) such that virtually all superoxide radicalsgenerated in mitochondria are converted to hydrogen peroxide whilechanneling 2% of total mitochondrial oxygen consumption, via superoxide,into the formation of H₂O₂ (Han et al., 2001; Boveris et al., 1972).

Superoxide and hydrogen peroxide are considered the primary reactiveoxygen metabolites. All other radicals are generated by way of secondaryreactions of these initially formed reactive oxygen metabolites(Eberhardt, 2001). Within mitochondria superoxide, therefore, is anintermediary in the formation of hydrogen peroxide.

Hydrogen peroxide is unique among reactive oxygen metabolites. It is nota radical, as it has no unpaired electrons however; it is considered aROM because it is the immediate precursor of the most damaging andchemically reactive radical known which is the hydroxyl radical (.OH).Hydrogen peroxide can undergo a one-electron reduction to form hydroxylradical. The reducing agent (electron donating species) can be atransition metal ion (Fenton reaction) or the superoxide radical(Haber-Weiss reaction).

Both iron and copper ions (present in tissues) can act as reducingagents in a Fenton reaction (Fe⁺²+H₂O₂→Fe⁺³+HO⁻+HO.) or(Cu⁺+H₂O₂→Fe⁺3+HO⁻+HO.) in the homolytic fission of hydrogen peroxide tohydroxyl radical and a hydroxide anion.

The Haber-Weiss reaction (O₂ ⁻.+H₂O₂→O₂+HO⁻+HO.) can also be acceleratedin vivo when iron is present in an iron catalyzed Haber-Weiss reaction(O₂ ⁻.+Fe⁺³→O₂+Fe⁺²) followed by the classic Fenton reaction above(Eberhardt, 2001). Superoxide, in addition to being generated within thecell, is also released to the extracellular compartment from varioussources including fibroblasts, endothelial cells and intestinal bacteria(O'Donnell et al., 1996; Souchard et al., 1998; Huycke et al., 2002;Huycke and Moore, 2002; Huycke et al., 2001). In biological systems,however, the iron catalyzed Haber-Weiss reaction is considered the majormechanism by which the highly reactive hydroxyl radical is generated(Kehrer, 2000).

The hydroxyl radical is an extraordinarily powerful oxidizing agent,which attacks other molecules at diffusion-limited rates and willindiscriminately destroy everything it encounters (Eberhardt, 2001;Fridovich, 1998; Chen and Schopfer, 1999). The hydroxyl radical is themost chemically reactive oxygen species formed in cellular metabolismand is principally responsible for the cytotoxic effects of oxygen inanimals (Chen and Schopfer, 1999). Despite its immensely damagingbiological effects the hydroxyl radical is continuously produced withrelative ease (Fridovich, 1998). This no doubt is due to theconstitutive nature of its precursor (H₂O₂), the ubiquitous distributionof transition metal catalyst (iron and copper) necessary for itsgeneration and the abundance of superoxide radical serving as theinitial electron donating (reducing) species.

Reacting at diffusion controlled rates means that hydroxyl radical willreact at every collision each time it encounters another molecule.Because of its extreme reactivity the hydroxyl radical will react withmost molecules in a site specific manner via addition and abstractionand, therefore, most molecules serve as scavengers of hydroxyl radical(Eberhardt, 2001).

Molecules interacting with hydroxyl radicals sustain severe damage tothe extent that the hydroxyl radical is able to crack polysaccharides,nucleic acids, and proteins located just a few atomic diameters(nanometers) from its site of generation (Chen and Schopfer, 1999).

The diffusion limited reaction rate of hydroxyl radical gives it theshortest half-life of any reactive oxygen metabolite (one nanosecond)(Kehrer, 2000). This extremely short reaction time makes the hydroxylradical very difficult to scavenge with any specific antioxidantmolecule. Detoxification of hydrogen peroxide, the immediate precursorto hydroxyl radical, therefore is crucial to normal cellular functionand survival. Consequently, very sophisticated intracellular enzymaticantioxidant mechanisms are in place to neutralize hydrogen peroxide atits site of generation before it can accumulate within cellularcompartments. These H₂O₂ neutralizing antioxidant enzymes are catalase(E.C. 1.11.1.6) and glutathione peroxidase (E.C. 1.11.1.9). The factthat there are two enzyme systems for H₂O₂ neutralization suggests thatremoval of hydrogen peroxide is essential for survival of the cell.

Catalase is located mainly within peroxisomes while glutathioneperoxidase is found throughout the cytoplasm and mitochondria(Eberhardt, 2001, pg 286; Davies, 2000; Cadenas and Davies, 2000). Thecompartmentalization of catalase coupled with a lower Km for H₂O₂ and afirst order catalytic reaction which is strictly proportional to theH₂O₂ concentration suggests that glutathione peroxidase is moreimportant for the removal of H₂O₂ than catalase (Eberhardt, 2001, pg.125).

A degradation profile for H₂O₂ has been established in human Jurkat Tcells. This study determined that glutathione peroxidase activity isresponsible for 91% of H₂O₂ consumption while catalase only contributesa minor role at 9% (Boveris and Cadenas, 2000). The relative importanceof these enzymes is manifested by the consequences of their respectivedeficiency states. Acatalasemia in humans is a relatively benign diseaseand most patients with this condition have no serious pathology.

Experimental acatalasemic mice likewise have no spontaneous healthproblems (Eaton and Ma, 1995). Complete absence of glutathioneperoxidase, in contrast, has not been reported in humans, presumablybecause the lack of this crucial enzyme precludes embryogenesis.

On a populational level, ethnic variation of glutathione peroxidase hasbeen recorded with individuals of Jewish or Mediterranean originexhibiting lower activities (The Metabolic and Molecular Basis ofInherited Disease, 2001, 8th ed., p. 4650). A two to four fold increasein incidence and prevalence of ulcerative colitis has also been reportedfor these ethnic groups (Roth et al., 1989).

As can be understood from the above, there remains a need in the art fortherapeutic modalities to treat inflammatory bowel diseases such asulcerative colitis.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns materials and methods for the preventionand treatment of disease conditions associated with oxidative stress ora compromised reducing environment. One aspect of the invention concernsmethods and compositions for treatment of inflammatory bowel disorders,such as Crohn's disease and ulcerative colitis. In one embodiment, atherapeutic composition of the present invention comprises a reducingagent. The reducing agent can be an agent capable of functioningintracellularly or it can be an extracellularly active agent or both. Inan exemplified embodiment, the reducing agent is 5-aminosalicylic acid(5-ASA) and/or sodium thiosulfate and/or dihydro lipoic acid and/oralpha lipoic acid (ALA) and/or pyruvate. In one embodiment, atherapeutic composition of the invention comprises sodium thiosulfate,bismuth subgallate, vitamin E, and sodium cromolyn. In an exemplifiedembodiment, a therapeutic composition is provided in a form suitable foradministration as a retention enema. In another embodiment, atherapeutic composition is provided in a form suitable for oraladministration.

Another aspect of the subject invention concerns compositions formulatedfor administration as an enema. An enema formulation of the inventioncomprises 5-ASA and a steroid compound. Steroid compounds contemplatedwithin the scope of the invention include corticosteroids. In oneembodiment, a composition suitable for administration as an enemacomprises an effective amount of 5-ASA and budesonide. In anotherembodiment, a composition of the invention comprises 5-ASA and ahydrocortisone, such as CORTENEMA. Other corticosteroids that can beused in the present invention include, but are not limited to,prednisone and prednisolone.

The subject invention also concerns methods of treating a person oranimal having an inflammatory bowel disorder. In one embodiment of thesubject methods, a person or animal in need of treatment is administereda composition of the present invention in a biologically compatible formor composition. In one embodiment, a composition to be administeredcomprises a reducing agent such as 5-ASA, sodium thiosulfate, dihydrolipoic acid (R-dihydro lipoic acid, L-dihydro lipoic acid, or both)and/or alpha lipoic acid or ALA. An enema formulation of the inventioncomprises 5-ASA and a steroid compound. Steroid compounds contemplatedwithin the scope of the invention include corticosteroids. In oneembodiment, a composition suitable for administration as an enemacomprises an effective amount of 5-ASA and budesonide. In anotherembodiment, a composition of the invention comprises 5-ASA and ahydrocortisone, such as CORTENEMA. Other corticosteroids that can beused in the present invention include, but are not limited to,prednisone and prednisolone. In an exemplified embodiment, thetherapeutic composition is administered rectally. In another embodiment,a composition for oral administration comprises alpha-lipoic acid,N-acetyl-L-cysteine (N-A-C), and L-glutamine.

The subject invention also concerns compositions formulated for oraladministration. In one embodiment, a composition comprises alpha-lipoicacid, N-acetyl-L-cysteine (N-A-C), and L-glutamine. The alpha-lipoicacid can be racemic alpha-lipoic acid, R-lipoic acid, orR-dihydro-lipoic acid. A composition of the present invention can alsocomprise compounds that inhibit tissue necrosis factor (TNF).

The subject invention also concerns kits and containers comprising atherapeutic composition or compounds of the present invention. Thecontainers can be selected for ease of administration of a therapeuticcomposition to a person or animal.

The subject invention also concerns methods and kits for detecting anddiagnosing an inflammatory bowel disorder, such as ulcerative colitis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show sigmoidoscopy results: distal sigmoid (FIG. 1A); distalsigmoid (FIG. 1B); mid-sigmoid (FIG. 1C); distal sigmoid (FIG. 1D); andrectum (FIG. 1E).

FIGS. 2A-2C show sigmoidoscopy results: distal sigmoid (FIG. 2A); rectum(FIG. 2B), and rectum (FIG. 2C).

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns materials and methods for the preventionand treatment of disease conditions associated with oxidative stress ora compromised reducing environment. One aspect of the invention concernsmethods and compositions for treatment of inflammatory bowel disorders,such as Crohn's disease and ulcerative colitis, and irritable boweldisorder. It has been discovered that the production of hydrogenperoxide (H₂O₂) and its overproduction and escape from cells of thegastrointestinal tract is a causal component of inflammatory boweldisorders such as ulcerative colitis. The cells of the body areconstantly producing oxygen radicals, including hydrogen peroxide, as aby-product of metabolism. These radicals must be neutralized within thecells before they can damage intracellular structures and lead to celldeath. The constant generation of oxygen radicals and hydrogen peroxideis an oxidative stress that is neutralized by the reducing capacity ofthe cell. The most important reducing substance within cell isglutathione. Thus, the reducing environment that cells require in orderto function is maintained by a delicate balance between the reductioncapacity of the cell (mainly glutathione) and oxygen radicals (mainlyhydrogen peroxide). Under normal conditions the intracellular hydrogenperoxide concentration is maintained very low by the constant productionof glutathione which neutralizes (reduces) H₂O₂ When this balance isdisturbed either by increased H₂O₂ generation, decreased glutathioneproduction or both, then hydrogen peroxide will accumulate within cellsand diffuse through the cellular membrane to the extracellular space.When this occurs in the rectal tissues, the colonic barrier to luminalbacterial products is disrupted allowing tissue penetration of antigenicmaterial into the normally sterile colonic lamina propria. Thesubsequent infiltration of activated neutrophils results in secretion ofenormous amounts of tissue destructive cytokines and additional oxygenradicals including hydrogen peroxide. Thus, a vicious cycle is set upwhereby cryptal neutrophils, upon exposure to fecal material, arestimulated to produce destructive cytokines and oxygen radicals in anattempt to rid the local environment of bacteria, which further damagesthe colonic tissue barrier thereby allowing additional bacterialinfiltration with amplification of the immune response and so on untilthe entire colon is involved. The production of hydrogen peroxide byinfiltrating neutrophils is able to diffuse into adjacent normal colonictissue, overwhelming its reducing capacity and causing oxidative damageto adjacent colonic barrier function and epithelial cells. This resultsin a contiguous spread of inflammation from the point of origin in therectum to the rest of the colon. Hydrogen peroxide that has diffused orescaped from a cell can be converted to a hydroxyl radical which cansubsequently disrupt cellular structures such as the basement membraneand tight junctions. This initiates the immune response which results inthe pathology associated with ulcerative colitis.

Ulcerative colitis can be divided into two phases. The first phase iscalled induction and begins with the extracellular diffusion of hydrogenperoxide to the extracellular environment. Throughout this phase theepithelial lining appears macroscopically intact and histologicallynormal. The damage is confined to molecular disruption of colonicepithelial tight junctions and basement membranes resulting intransitory increased colonic permeability to intestinal antigens. Thereis no rectal bleeding during this phase and this process may go on formonths to years resulting in sporadic extra intestinal manifestationssuch as myalgias, arthralgias and faciitis due to intermittent immuneactivation subsequent to transitory colonic sub-mucosal antigenicpenetration. Due to the high colonic epithelial turnover rate of aboutthree days it is possible to repair the damage and restore the colonicbarrier if the initial damage is not overwhelming. However, if theepithelial barrier cannot be reassembled and antigenic invasion issustained, then further immune activation in the form of neutrophilicinfiltration will occur.

The second phase of ulcerative colitis begins with neutrophilic invasioninto the colonic tissues and is called the propagation phase. It isduring this phase that neutrophil derived cytokines and oxygen radicalsinitiate tissue damage leading to mucosal ulceration and rectal bleedingand diarrhea characteristic of this disease.

The importance of two distinct phases of ulcerative colitis lies in theability to modify the inflammatory process during the induction phasevia manipulation of risk factors which can be positive(pro-inflammatory, increasing H₂O₂ production) or negative(anti-inflammatory, decreasing H₂O₂ production). The propagation phase,as the name implies, is self-sustaining and auto-stimulating, and is notaffected by risk factors. It is during the propagation phase thatindividuals manifest rectal bleeding due to colonic tissue destructionand without external intervention to reverse the process may developextensive colonic inflammation leading to colectomy. Colectomy willeliminate the source of induction and propagation along with thatsegment of intestine having a damaged and permeable barrier. This willabolish the portal of systemic antigenic entry and terminate theinflammatory process. However, colonic inflammation can be terminatedanytime during induction if risk factors promoting H₂O₂ production arerecognized and eliminated. Propagation can also be terminated byappropriate intervention to treat a patient with methods andcompositions of the present invention.

Any materials that can be used to neutralize hydrogen peroxide or itsdecomposition products (hydroxyl radical or hydroxide anion) arecontemplated within the scope of the invention. These include, but arenot limited to, oxidizing agents, reducing agents, enzymes such asglutathione peroxidase and catalase, catalysts (such as zinc dust orother metal powders or metal catalysts), manganese in a bicarbonatebuffer, and asbestos fibers or other fibers able to decompose hydrogenperoxide. Glutathione, or its precursor amino acids (glycine, cysteineand glutamate) can also be used in the compositions and methods of theinvention. Monoester or diester glutathione derivatives can be used withthe subject invention as glutathione with ester groups attached is takenup into cells more readily than glutathione.

In one embodiment, a therapeutic composition of the present inventioncomprises at least one reducing agent. The reducing agent can be onethat acts primarily extracellularly, primarily intracellularly, or bothextracellularly and intracellularly. In one embodiment, a therapeuticcomposition of the invention comprises at least one extracellularreducing agent and at least one intracellular reducing agent.

In an exemplified embodiment, the reducing agent is a thiosulfate ion,which can be provided in the form of a salt such as, for example, sodiumthiosulfate, ammonium thiosulfate, calcium thiosulfate, potassiumthiosulfate, silver thiosulfate, choline thiosulfate, gold sodiumthiosulfate, magnesium thiosulfate hex hydrate, and thiosulfatehyposulfite. Examples of other reducing agents contemplated within thescope of the invention include, but are not limited to, metalborohydrides, sodium hydrosulfite, dimethylthiourea, sodium bisulfite,thiourea dioxide, diethylhydroxylamine, zinc dust, sodiumcyanoborohydride, sodium hydride, trimethyl borate, benzyltriphenphosphonium chloride, butyl triphenphosphonium bromide, ethyltriphenphosphonium acid acetate, ethyl triphenphosphonium bromide, ethyltriphenphosphonium iodide, ethyl triphenphosphonium phosphate, andtetrabutyl phosphonium acid acetate. Another reducing agent that can beused is glutathione, or a monoester or diester glutathione, diesterglutathione or multiester glutathione derivative.

In an exemplified embodiment, an intracellular reducing agent of theinvention is alpha lipoic acid (ALA), dihydro lipoic acid, or pyruvate.Both of these are capable of entering a cell and reacting with H₂O₂. ALAis a small molecule (MW 206.3, CAS #1077-28-7). It is known by a varietyof names which vary depending upon its redox state (oxidized or reduced)and the enantiomeric configuration around the number three carbon chiralcenter (*). The older relative (comparison) based D and L nomenclaturehas been replaced by the designation R and S indicating absolutestereochemical configuration.

Alternative terminology for Alternative terminology for Alpha-lipoicacid Dihydro-lipoic acid Thioctan 6,8 thioctic acid Thioctic acid DL-6-Thioctic acid 1,2 dithiolane-3-valeric acid R-Dihydro-lipoic acid 1,2dithiolane-3-pentanoic acid 6,8 dithiooctanoic acidDL-1,2-Dithiolan-3-valeriansaeure RS-Dihydro-lipoic acid

ALA is an eight carbon cyclic disulfide containing fatty acid which issynthesized in trace amounts within mitochondria in all cells of thebody. Only the R-isomer is synthesized naturally. In its natural stateALA is covalently bonded, via its terminal carboxyl in an amide linkage,to the epsilon amino group of lysine residues which form part ofmulti-subunit enzyme complexes that catalyze vital energy metabolismreactions within mitochondria. There is very little free ALA in thecytoplasm or circulation.

The bonding of ALA to its cognate protein is accomplished as apost-translational modification of the enzyme. In its protein boundstate it is a required enzymatic co-factor called lipoamide. The enzymecomplexes which use ALA are the pyruvate dehydrogenase complex whichcatalyzes the conversion of pyruvate to acetyl-CoA, a vital substratefor energy production via the Krebs (citric acid) cycle. Thealpha-ketoglutarate complex which catalyzes another important Krebscycle reaction, the branched chain alpha-keto acid dehydrogenase complexwhich catalyzes the oxidative decarboxylation of three branched chainamino acids (valine, leucine and isoleucine) generating acetyl-CoA forentry into the Krebs cycle and finally the glycine cleavage systemcomplex that catalyzes the formation of 5,10 methylene tetrahydrofolatewhich plays a vital role in the synthesis of nucleic acids.

The natural function of ALA is to bind and transfer acyl groups tosuccessive enzymatic active sites among the subunits of each enzymecomplex. In this process of acyl transfer ALA is reduced todihydrolipoic acid and subsequently re-oxidized back to ALA by itsattached cognate enzyme which readies it for the next acyl transfer. ALAhas a high degree of bioavailability after oral administration andexhibits both lipid and water solubility. This allows its distributionto both intra and extra cellular compartments.

A therapeutic composition of the present invention can optionallyinclude one or more of the following:

-   -   1) a compound or composition that is antibacterial or that        prevents or inhibits adherence of bacteria to gastrointestinal        tissues or cells. In one embodiment, the compound can be a        bismuth salt. In an exemplified embodiment, the compound can be        bismuth subgallate. Antibiotics active against bacteria present        in the gastrointestinal tract can be included. In a preferred        embodiment, the compound is active against Bacteroides.    -   2) a compound or composition that adds viscosity (for steric        hindrance) and/or that inhibits epithelial lipid peroxidation,        such as, for example, d-alpha-tocopherol (vitamin E),        carboxymethylcellulose or other viscous mono or polysaccharide        compounds (e.g., honey).    -   3) a compound or composition that inhibits mast cells and/or        that helps to seal or repair tight junctions between cells in        the gastrointestinal tract. In one embodiment, the compound can        be sodium cromolyn.    -   4) a compound or composition that scavenges hydroxyl radicals.        In one embodiment, the compound can be dimethyl sulfoxide        (DMSO), mannitol, methional, deoxyribose or DMPO        (5,5-dimethylpyrollidine-N-oxide).    -   5) a compound or composition that inhibits or blocks NADPH        oxidase, such as DMSO or apocynin.    -   6) a compound or composition that kills or inhibits colonically        localized neutrophils, or that prevents or inhibits neutrophils        from entering the colon or exiting the colonic vasculature. In        one embodiment, antibodies or other blocking agents of vascular        adhesion molecules (ICAMS) present on vascular endothelium, such        as selectin, or antibodies or other blocking agents of the        corresponding neutrophilic counter ligand, such as integrin, can        be used.    -   7) a compound or composition that stops or inhibits neutrophils        from producing hydrogen peroxide. Examples include DMSO and        Trental.    -   8) a compound or composition that chelates or sequesters iron        molecules that are necessary for the reduction of hydrogen        peroxide to a hydroxyl radical. In one embodiment, the iron        chelating agent Desferal (Deferoxamine) can be used.    -   9) the compound 5-aminosalicylic acid (5-ASA) or colazal        (balsalazide disodium) can be included.    -   10) a compound that neutralizes or scavenges hydroxide ions. In        one embodiment, a composition of the invention can comprise a        weak acid or weak base, such as in the form of a buffered        solution at a pH of from about 6.8 to about 7.4 comprising        sodium bicarbonate.    -   11) any agent or therapy that will inhibit the electron        transport chain or any of its components (e.g., an agent or        therapy that suppresses cytochrome oxidase enzyme).

In one embodiment, a composition of the invention comprises a reducingagent and an NADPH-oxidase inhibitor. Preferably, the reducing agent isa thiosulfate salt, such as sodium thiosulfate, and the NADPH-oxidaseinhibitor is apocynin. In a preferred embodiment, the composition isprovided in an orally administered capsule that delays dissolving untilit is present in the colon.

The compounds of the invention can be administered as a singlecomposition, or they can be administered individually at the same ordifferent times and via the same or different route (e.g., oral, rectal,etc.) of administration. In one embodiment, a composition of theinvention is provided in a mixture or solution suitable for rectalinstillation and comprises sodium thiosulfate, bismuth subgallate,vitamin E, and sodium cromolyn. In one embodiment, a therapeuticcomposition of the invention comprises, in a suppository form, butyrate,and glutathione monoester, glutathione diethylester or other glutathioneester derivatives. The suppository can optionally include sodiumthiosulfate and/or vitamin-E.

The subject invention concerns methods for preventing and/or treatingdiseases that are caused or exacerbated by oxidative stress or that arecaused by or exacerbated by a compromised intracellular or extracellularreducing environment. One embodiment of the subject invention concernsmethods of preventing and/or treating a person or animal having aninflammatory bowel disorder, such as, for example, ulcerative colitis.The subject invention also concerns methods for preventing and/ortreating radiation induced proctitis that can result from radiationtreatment of prostate cancer and other disease conditions. Other diseaseconditions that can be prevented and/or treated using methods andcompositions of the present invention include, but are not limited to,Parkinson's disease, cataracts, cerebral palsy, and gastrointestinalcancers, such as stomach and colon cancers. Compositions of the presentinvention can also be used to treat or prevent pouchitis after acolectomy. Pouchitis is an inflammation of the “J” pouch that issurgically constructed from the last foot of small intestine after thecolon is taken out. The last six inches of small intestine is bent upand sewn back onto the small intestine. The hole is made at the bend ofthe “J” and that is connected to the anus. The purpose is to have areservoir for stool to avoid the external bag.

The small intestine is more permeable to luminal contents and havingstool stored in it in that fashion can lead to inflammation due to theincreased bacterial load and increased oxidative stress. Compositionsformulated for administration as an enema or oral administration can beused to treat or prevent pouchitis.

The subject invention also concerns methods for treating hemorrhoids. Inone embodiment, a liquid enema formulation of the invention is applieddirectly to the hemorrhoid. In a specific embodiment, a liquid enemaformulation of the invention is placed on a suitable material such asgauze or an absorbent pad and the enema containing material is contactedwith the hemorrhoid, typically for about 2 to 4 hours. This applicationis typically performed once or twice daily.

In one embodiment of the subject methods, a person or animal in need oftreatment is administered an effective amount of a therapeuticcomposition of the present invention in a biologically compatible formor composition. For preventative therapy, an effective amount of atherapeutic composition of the invention is administered to a person oranimal prior to onset of the condition to be treated. For example, aneffective amount of a therapeutic composition of the invention isadministered prior to radiation treatment of prostate cancer in order toprevent radiation induced proctitis. In an exemplified embodiment, thereducing agent of the composition is ALA and/or dihydro lipoic acidand/or pyruvate and/or 5-ASA and/or a thiosulfate salt such as, forexample, sodium thiosulfate. In one embodiment, the therapeuticcomposition is administered rectally or by delayed dissolving oralcapsule. Delayed dissolution dosage forms include pH-dependent capsulesand coatings that only dissolve at the pH associated with the colonicenvironment. Examples of pH-dependent materials include, but are notlimited to, methyl methacrylate, methacrylic acid and/or ethyl acrylatepolymers, including for example, ammonio methacrylate copolymer. Otherdosage forms for delivery of a composition of the invention to the coloninclude, for example, time-dependent delivery systems,pressure-dependent delivery systems, bacterial-dependent systems (Basitet al., 2003). Also contemplated are dosage forms that utilize oxidationpotential-dependent systems. Colon content has a much higher oxidativepotential than the small intestinal contents since many times morebacteria are present in the colon. An oxidation potential-dependentsystem is a delivery system that is sensitive to oxidation potential andreleases its contents or becomes active when the capsule is exposed tothe higher oxidation level in the colon. This can be in the form of aprodrug which is degraded to the active drug when oxidized and capsuleswhich dissolve when exposed to the high level of oxidation in the colon.In one embodiment, a combination of rectally and orally administeredcompositions are given to a patient, particularly if the patient hasrectal bleeding. After bleeding is controlled, rectal therapy canoptionally be discontinued and oral therapy maintained as necessary.Thiosulfates, such as sodium thiosulfate can optionally be administeredin a solution intravenously, e.g., solutions of from about 10% to about25% (w/v) sodium thiosulfate can be given. Methods of the presentinvention also contemplate institution of lifestyle changes of thepatient, as described herein, either alone or in conjunction with theuse of therapeutic compositions of the invention.

Dosage ranges for the various compounds to be administered to anindividual patient can be determined by an ordinarily skilled clinician.Examples of dosage ranges provided herein are for guidance and shouldnot be construed as limiting the scope of the invention in regard todosages that can be administered. Dosage ranges can be, for example:

sodium thiosulfate: 150-250 mg/kg body weight alpha lipoic acid 10-20mg/kg body weight dihydro lipoic acid 10-20 mg/kg body weight pyruvate50-100 mg/kg body weight bismuth subgallate: 2-4 mg/kg body weightvitamin E: 25-30 IU/kg body weight cromolyn sodium: 1-3 mg/kg bodyweight

In one embodiment, the components sodium thiosulfate, bismuthsubgallate, vitamin E, and cromolyn sodium can be prepared in aretention enema in sterile water according to the following:

-   -   Step 1: Dissolve sodium thiosulfate in water by gently shaking        until all crystals are dissolved.    -   Step 2: Add cromolyn sodium until completely dissolved.    -   Step 3: Add bismuth subgallate and gently shake until completely        suspended.    -   Step 4: Add vitamin E and shake until suspended.

The methods of the present invention also include oral administration ofa drug that lowers endogenous catecholamines, such as clonidine, wheresuch treatment is indicated by the symptoms and risk factors presentedby the patient. Monoamine oxidase (MAO) inhibitors that inhibit orprevent mitochondrial MAO from metabolizing endogenous catecholaminescan also be administered as part of a patient treatment regimen and iscontemplated within the scope of the present invention. Preferably, theMAO inhibitor is one that does not pass through the blood-brain barrier.In one embodiment of the invention, a combination of clonidine (or asimilar drug) and an MAO inhibitor is administered to a patient.

Also contemplated within the scope of the invention is theadministration of NADPH-oxidase inhibitors, such as Trental(pentoxifylline) and apocynin; these can be administered as a delayeddissolving oral capsule that dissolves in the colon, or as a rectalsolution. Pentoxifylline has anti-inflammatory activity and may functionas a purinergic agonist via an adenosine receptor on the surface of theinfiltrating neutrophil which can inhibit NADPH oxidase and apoptosis.This oral therapy can be continued, along with lifestyle changes, asmaintenance therapy to prevent re-induction and relapse.

In one embodiment, a composition of the invention comprises a reducingagent and an NADPH-oxidase inhibitor. In one embodiment, the reducingagent is a thiosulfate salt, such as sodium thiosulfate, and/or 5-ASAand/or ALA and/or dihydro lipoic acid and/or pyruvate, and theNADPH-oxidase inhibitor is apocynin. In one embodiment, the compositionis provided in an orally administered form, such as a capsule, thatdissolves in a subject's stomach and/or small intestine. In anotherembodiment, the composition is provided in an orally administeredcapsule that delays dissolving until it is present in the colon. Delayeddissolution dosage forms include pH-dependent capsules and coatings thatonly dissolve at the pH associated with the colonic environment.Examples of pH-dependent materials include, but are not limited to,methyl methacrylate, methacrylic acid and/or ethyl acrylate polymers,including for example, ammonio methacrylate copolymer. Other dosageforms for delivery of a composition of the invention to the coloninclude, for example, time-dependent delivery systems,pressure-dependent delivery systems, bacterial-dependent systems (Basitet al., 2003). Also contemplated are dosage forms that utilize oxidationpotential-dependent systems.

Another aspect of the subject invention concerns compositions formulatedfor administration as an enema. An enema formulation of the inventioncomprises a reducing agent (or any other agent having a similar mode ofaction) and a steroid. In one embodiment, an enema formulation of theinvention comprises an aminosalicylic acid, such as 5-ASA(5-aminosalicylic acid; also known as mesalamine), or 4-ASA(4-aminosalicylic acid), or any analog or derivative of a salicylicacid, and a steroid compound. In another embodiment, the compositioncomprises sulfasalazine (Azulfidine) as the reducing agent. Steroidcompounds contemplated within the scope of the invention includecorticosteroids. In an exemplified embodiment, the steroid comprisesbudesonide, or an analog or derivative thereof. In a specificembodiment, a composition suitable for administration as an enemacomprises an effective amount of 5-ASA and budesonide. In anotherspecific embodiment, a composition of the invention comprises 5-ASA anda hydrocortisone compound, such as CORTENEMA. Other corticosteroids thatcan be used in the present invention include, but are not limited to,prednisone, prednisolone, betamethasone, beclometasone, and tixocortol.The enema formulation can optionally comprise polysorbate-80 (or anyother suitable emulsifying agent), and/or any short chain fatty acid(e.g., a five, four, three, or two carbon fatty acid) as a colonicepithelial energy source, such as sodium butyrate (4 carbons),proprionate (3 carbons), acetate (2 carbons), etc., and/or any mast cellstabilizer, such as cromolyn sodium (GASTROCROM) or Nedocromil sodium(ALOCRIL). In a further embodiment, an enema formulation of theinvention can comprise ALA. In one embodiment, the ALA is provided as aracemic mixture of the R and L isomers of ALA. In another embodiment,the ALA is provided as R-alpha-lipoic acid. In another embodiment, theALA is provided as R-dihydro lipoic acid. In one embodiment, an enemaformulation of the invention comprises an effective amount of 5-ASA,budesonide, sodium butyrate, cromolyn sodium, and optionally analpha-lipoic acid, such as R-dihydro lipoic acid.

In one embodiment, the composition comprises from about 200 mg to about3000 mg, or about 500 mg to about 1,500 mg of 5-ASA, or from about 750mg to about 1,250 mg of 5-ASA. In a specific embodiment, the compositioncomprises about 1,150 mg of 5-ASA. In another embodiment, thecomposition comprises about 2,600 mg of 5-ASA. In one embodiment, 5-ASAis applied at 30-40 mg/kg body weight. The composition can also comprisefrom about 1 mg to about 10 mg of budesonide or hydrocortisone or about8 mg of hydrocortisone, or from about 2.5 mg to about 7.5 mg ofbudesonide or hydrocortisone or about 8 mg of hydrocortisone. In aspecific embodiment, the composition comprises about 5 mg of budesonide.In one embodiment, budesonide is applied at 0.05-0.15 mg/kg body weight.If the composition comprises cromolyn sodium it can be present in anamount from about 10 mg to about 200 mg, or from about 20 mg to about100 mg, or from about 30 mg to about 70 mg. In a specific embodiment,cromolyn sodium is provided in an amount of about 40 mg. In anotherembodiment, the cromolyn sodium is provided in an amount of about 100mg. If the composition comprises polysorbate-80, it can be provided at aconcentration from about 1% (v/v) to about 10% (v/v). If the compositioncomprises sodium butyrate it can be present in an amount of about 500 toabout 1500 mg. In one embodiment, sodium butyrate is applied at 4-6mM/kg body weight. In a specific embodiment, polysorbate-80 is providedin the composition at a concentration of about 6% (v/v). In anexemplified embodiment, a composition suitable for administration as anenema is formulated as follows: 17 cc of 5-ASA (about 1,150 mg of5-ASA), 1 cc of budesonide (at 5 mg per cc), 2 cc of cromolyn sodium (at20 mg per cc), and polysorbate-80 at 6% (v/v). In another specificembodiment, a composition comprises 10 cc of hydrocortisone (16.6 mgtotal), 30 cc of 5-ASA (2 gm total), and optionally alpha-lipoic acidand/or L-glutamine and/or N-acetyl cysteine and/or 5 cc cromolyn sodium(100 mg), and/or 12.5 cc sodium butyrate (1.1 gm).

In a specific embodiment, an enema formulation of the inventioncomprises 5-ASA, budesonide, cromolyn sodium, and sodium butyrate. Theformulation can comprise, for example, about 1 to 5 grams of 5-ASA;about 1 to 10 mg of budesonide; about 10 to 1000 mg of cromolyn sodium;and about 5 to 50 millimoles of sodium butyrate. In a more specificformulation, the enema can be prepared to comprise about 2.7 grams of5-ASA (40 cc of a 60 cc bottle of 5-ASA containing 4 grams of 5-ASA inthe 60 cc); about 5 mg budesonide (in 1 cc); about 100 mg cromolynsodium (in 5 cc); and about 15 millimoles of sodium butyrate (15 cc of a1 molar solution of sodium butyrate in water).

Enema compositions of the invention can be administered to a patient ondosage and schedule as determined by a clinician. In one embodiment, anenema of the invention can be administered daily. Optionally, it can bealternated every other day with an enema that contains the reducingagent, e.g., 5-ASA, and optionally the cromolyn sodium and/orpolysorbate-80 but without the steroid. In one embodiment, a patient isgiven an enema of the invention daily for approximately a month.Optionally, in the second month of treatment, the amount of steroid inthe enema is reduced over time; for example, the steroid can be reducedover four weeks of treatment until no steroid is administered in theenema (e.g., at the start of the third month of treatment). In the thirdmonth of treatment, an enema of the invention to be administered dailyto a patient may comprise the reducing agent (e.g., 5-ASA), and a shortchain fatty acid (e.g., sodium butyrate) but omitting the steroid. In afourth month of treatment, an enema of the invention to be administereddaily to the patient may comprise the reducing agent but omit the shortchain fatty acid and the steroid. In a fifth month of treatment, apatient may receive the same enema as in the fourth month but everyother day or, optionally, on an as needed basis.

The subject invention also concerns compositions formulated for oraladministration. These oral compositions can be administered as aseparate treatment or in conjunction with enema formulations of theinvention. In one embodiment, compositions of the invention areadministered orally during enema treatment, and continue after cessationof enema treatment. In one embodiment, a composition comprises an oralreducing agent such as an alpha-lipoic acid or any oral agent whichdirectly, or indirectly through an intermediary mechanism, acts as anintracellular or extracellular reducing agent and neutralizes orotherwise prevents the formation of intra or extracellular hydrogenperoxide or oxygen radicals. Other oral reducing agents contemplatedwithin the scope of the invention include, but are not limited to,sodium thiosulfate, mercaptopropionylglycine, N-acetylcysteine,glutathione, melatonin, CoQ 10, Ebselen, and an aminosalicylic acid suchas 5-aminosalicylic acid (5-ASA). 5-ASA is also available as an oralformulation (in addition to an enema formulation) (including, forexample, Azulfidine, Asacol, Dipentum, mesalamine, Balsalazide,Olsalazine) and is capable of neutralizing extracellular peroxide andoxygen radicals. The alpha-lipoic acid can be racemic alpha-lipoic acid,R-lipoic acid, R-dihydro-lipoic acid, L-lipoic acid, and/orL-dihydro-lipoic acid. In one embodiment, the apha-lipoic acid isadministered at 5-10 mg/kg. In a specific embodiment, about 100 mg to1000 mg or more of alpha-lipoic acid, preferably as the R-dihydro-lipoicacid, is orally administered daily to a patient. In a more specificembodiment, about 600 mg of alpha-lipoic acid, preferably as theR-dihydro-lipoic acid, is orally administered daily to a patient;optionally, the patient can receive about 300 mg of the alpha-lipoicacid, preferably as the R-dihydro-lipoic acid, twice daily.

In one embodiment, an oral composition of the invention comprisesalpha-lipoic acid, and/or N-acetyl-L-cysteine (N-A-C), and/orL-glutamine. A composition of the present invention can also comprisecompounds that inhibit tissue necrosis factor (TNF). TNF inhibitorycompounds contemplated within the scope of the invention includeresveratrol, stinging nettle leaf extract, and berberine. A compositionof the present invention can also optionally include compounds thatdirectly neutralize H₂O₂ (e.g., calcium pyruvate), compounds that helpprotect the proteins from oxidation and degradation (e.g., L-carnosine),and/or compounds that are protective of nucleic acids (e.g., calciumD-glucarate). The composition can also optionally comprise one or moreof the following: selenium, vitamin B-2 (riboflavin), vitamin B-12,folic acid, and biotin. In a specific embodiment, a composition of theinvention comprises 300 mg of R-dihydro-lipoic acid, 500 mg N-A-C, 500mg L-glutamine, 200 μg selenium, 100 mg vitamin B-2, 500 μg vitaminB-12, 800 μg folic acid, 2,000 μg biotin, 1,500 mg calcium pyruvate, 150mg resveratrol, 275 mg stinging nettle leaf extract, and 200 mg rawstinging nettle leaf powder, berberine in the form of golden seal rootcomplex 150 mg and golden seal powder 300 mg, 1,000 mg L-carnosine, and250 mg calcium D-glucarate.

In one embodiment, the composition is provided in an orally administeredform, such as a capsule, that dissolves in a subject's stomach and/orsmall intestine. In another embodiment, the composition is provided inan orally administered capsule that delays dissolving until it ispresent in the colon. Delayed dissolution dosage forms includepH-dependent capsules and coatings that only dissolve at the pHassociated with the colonic environment. Examples of pH-dependentmaterials include, but are not limited to, methyl methacrylate,methacrylic acid and/or ethyl acrylate polymers, including for example,ammonio methacrylate copolymer. Other dosage forms for delivery of acomposition of the invention to the colon include, for example,time-dependent delivery systems, pressure-dependent delivery systems,bacterial-dependent systems (Basit et al., 2003). Also contemplated aredosage forms that utilize oxidation potential-dependent systems.

The enema compositions and the oral compositions of the invention can beadministered in conjunction with changes in a patient's diet. Dietarychanges contemplated within the scope of the present invention includeincreased consumption of mineral oil, insoluble fiber, soluble fiber,prune juice, and VSL#3. It is preferable that alcohol be avoided in thediet of the patient being treated.

The methods and compositions of the present invention can be used withhumans and other animals. Compositions of the present invention can beadministered to an animal in need of treatment as described herein,i.e., orally, and/or as an enema, etc. The other animals contemplatedwithin the scope of the invention include domesticated, agricultural, orzoo- or circus-maintained animals. Domesticated animals include, forexample, dogs, cats, rabbits, ferrets, guinea pigs, hamsters, pigs,monkeys or other primates, and gerbils. Agricultural animals include,for example, horses, mules, donkeys, burros, cattle, cows, pigs, sheep,and alligators. Zoo- or circus-maintained animals include, for example,lions, tigers, bears, camels, giraffes, hippopotamuses, andrhinoceroses.

As noted above, the oral and enema compositions of the invention can beadministered independently or in conjunction together to a person oranimal in need of treatment. Thus, in one embodiment, a method of theinvention comprises administering an effective amount of only an oralcomposition of the invention, or an effective amount of only an enemacomposition of the invention, or an effective amount of both an oral andan enema composition of the invention to a person or animal in need oftreatment. In one embodiment, an effective amount of an oral compositioncomprising an ALA (such as R-dihydro-lipoic acid) and an effectiveamount of an enema composition comprising an aminosalicylic acid (suchas 5-ASA), and/or any suitable steroid (such as budesonide), and/or anymast cell stabilizer (such as cromolyn sodium), and/or any short chainfatty acid (such as sodium butyrate), and optionally an emulsifyingagent (such as polysorbate-80), are administered to the person oranimal. The oral and enema compositions can be administered daily, orevery other day or every other two days, or every other three days,etc., or weekly, or on any other schedule as determined to beappropriate by the ordinarily skilled artisan. The oral and enemacompositions can be administered on alternate days, e.g., oral on dayone, enema on day two, etc. In one embodiment, the oral composition isadministered every day, and the enema composition is administered everyother day, or every two days, or every three days, or every four days,or every five days, or every six days, or once a week, etc. The oral andenema compositions can also be administered independently one or moretimes (e.g., two times, three times, etc.) per day.

Compounds useful in the subject invention can be formulated according toknown methods for preparing pharmaceutically useful compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin describesformulations which can be used in connection with the subject invention.In general, the compositions of the subject invention will be formulatedsuch that an effective amount of the compound is combined with asuitable carrier in order to facilitate effective administration of thecomposition. The compositions used in the present methods can also be ina variety of forms. These include, for example, solid, semi-solid, andliquid dosage forms, such as tablets, pills, powders, liquid solutionsor suspension, suppositories, injectable and infusible solutions, andsprays. The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions alsopreferably include conventional pharmaceutically acceptable carriers anddiluents which are known to those skilled in the art. Examples ofcarriers or diluents for use with compounds include ethanol, dimethylsulfoxide, glycerol, alumina, starch, and equivalent carriers anddiluents. To provide for the administration of such dosages for thedesired therapeutic treatment, new pharmaceutical compositions of theinvention will advantageously comprise between about 0.1% and 45%, andespecially, about 1 and 15% by weight of the total of one or more of thecompounds based on the weight of the total composition including carrieror diluent.

The subject invention also concerns dosage forms of the compounds andcompositions of the invention. In one embodiment, compounds andcompositions are provided in orally or rectally administered dosageforms. A dosage form for oral administration comprising a capsule thatdissolves in the colon and containing an effective amount of i) areducing agent, for example, 5-ASA and/or sodium thiosulfate and/or APAand/or dihydro lipoic acid and/or pyruvate and/or an alpha-lipoic acid,and optionally ii) an NADPH-oxidase inhibitor, for example, apocynin, isspecifically contemplated in the present invention. Delayed dissolutiondosage forms include pH-dependent capsules and coatings that onlydissolve at the pH associated with the colonic environment. Examples ofpH-dependent materials include, but are not limited to, methylmethacrylate, methacrylic acid and/or ethyl acrylate polymers, includingfor example, ammonio methacrylate copolymer. Other dosage forms fordelivery of a composition of the invention to the colon include, forexample, time-dependent delivery systems, pressure-dependent deliverysystems, bacterial-dependent systems (Basit et al., 2003). Alsocontemplated are dosage forms that utilize oxidation potential-dependentsystems.

The compounds of the subject invention can also be administeredutilizing liposome technology, slow release capsules, implantable pumps,biodegradable containers and other means known in the art. Thesedelivery vehicles and methods can, advantageously, provide a uniformdosage over an extended period of time.

The subject invention also concerns containers comprising a therapeuticcomposition of the present invention. The containers can be selected forease of storage and/or administration of a composition to a person oranimal, e.g., a container can be one suitable for use in rectaladministration of a therapeutic composition. The containers can becomposed of any suitable material, including glass, plastic, etc. andcan be disposable and/or recyclable. Therapeutic compositions of thepresent invention are preferably provided as a sterile composition in asterile container. The subject invention also concerns kits comprising,in one or more containers, a therapeutic composition or compound of theinvention. In one embodiment, a kit of the invention comprises, in oneor more containers, a reducing agent, and/or an aminosalicylic acid,and/or a steroid, and/or a short chain fatty acid, and/or an emulsifyingagent, and/or an antibacterial and/or antiadherence agent, and/or aviscosity enhancing and/or lipid peroxidation inhibitor, and/or a mastcell inhibitor and/or a mast cell stabilizer, and/or an agent thatrepairs, seals, or regenerates tight junctions between cells, e.g., anepidermal growth factor (EGF). In an exemplified embodiment, the kitcomprises the compounds sodium thiosulfate, bismuth subgallate, vitaminE, and sodium cromolyn. In another embodiment, a kit comprises 5-ASA,budesonide, cromolyn sodium, and sodium butyrate. The compounds of theinvention can be provided in a kit in a single pre-mixed dosage form, orin individual dosage units that are mixed together prior toadministration, or that are administered individually. In oneembodiment, a kit of the invention comprises compounds of an enemaformulation of the invention and materials or articles for effectingadministration of an enema.

The subject invention also concerns methods for screening for, assessingrisk of developing, and/or diagnosing conditions associated withoxidative stress, such as ulcerative colitis and other inflammatorybowel disorders, and Parkinson's disease. In one embodiment, a method ofthe invention comprises screening a person or animal for the presence ofSNPs (single nucleotide polymorphisms) in genes that code for enzymes inthe antioxidant network of enzymes that help maintain intracellularredox buffering capacity of cells, wherein the SNP(s) results in amutation that affects proper expression of the gene or gene product orthat results in a mutation that decreases or eliminates activity of theenzyme. Standard methods and materials for screening for SNPs are knownin the art. Enzymes included within the scope of the invention include,but are not limited to, glutathione-cysteine ligase, glutathionesynthetase, glutathione disulfide reductase, glutathione peroxidase,methylenetetrahydrofolate reductase, malic enzyme, glucose-6-phosphatedehydrogenase, and phosphogluconate dehydrogenase. In one embodiment, amethod of the invention comprises screening for or identifying in aperson or animal any gene encoding for the glutamate-cysteine ligaseenzyme (EC 6.3.2.2) or glutathione synthetase enzyme (EC 6.3.2.3) whichcontains an SNP that results in decreased enzymatic activity resultingin a diminished synthesis of glutathione. In one embodiment, a method ofthe invention comprises screening for or identifying in a person oranimal any gene encoding for the glutathione-disulfide reductase (EC1.8.1.7) enzyme which contains an SNP that results in decreasedenzymatic activity resulting in a diminished synthesis of NADPH. In oneembodiment, a method of the invention comprises screening for oridentifying in a person or animal any gene encoding for the glutathioneperoxidase (EC 1.11.1.9) enzyme which contains an SNP that results indecreased enzymatic activity resulting in a diminished neutralization ofhydrogen peroxide. In one embodiment, a method of the inventioncomprises screening for or identifying in a person or animal any geneencoding for the methylenetetrahydrofolate reductase (EC1.5.1.20) enzymethat results in inhibition of glutathione peroxidase (EC 1.11.1.9). Inone embodiment, a method of the invention comprises screening for oridentifying in a person or animal for the malic enzyme (EC 1.1.1.40),glucose-6 phosphate dehydrogenase (EC 1.1.1.49) or phosphogluconatedehydrogenase enzyme (EC 1.1.1.44) which contains an SNP that results indecreased enzymatic activity resulting in a diminished synthesis ofNADPH. Known SNPs for genes can be searched and identified at thewebsite:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=DisplayFiltered&DB=snp.

The subject invention also concerns methods for detecting and diagnosingulcerative colitis, and diagnostic kits for the same. Any means fordetecting or measuring a physiological parameter associated with theinduction phase or the propagation phase of ulcerative colitis asdescribed herein is contemplated within the scope of the presentinvention. In one embodiment, rectal tissue is screened for the presenceof increased levels of hydrogen peroxide, decreased levels ofglutathione, or both. Techniques and materials for detecting orquantitating hydrogen peroxide and glutathione are known in the art. Inanother embodiment, colonic tissue is screened for the infiltration ofactivated neutrophils and/or the presence of neutrophil-derivedcytokines or products of lipid peroxidation. Techniques and materialsfor detecting or quantitating activated neutrophils andneutrophil-derived cytokines are known in the art. Assays of theinvention can optionally include controls, both positive and negative,and/or other standards (e.g., normal rectal tissue or cells; standard ornormal concentration of hydrogen peroxide; standard or normalconcentration of glutathione, etc.) for evaluation with the results of atested sample. Assays screening for multiple physiological parametersassociated with ulcerative colitis are included within the scope of thepresent invention. Tissue to be used for the assays of the presentinvention can be local (e.g., rectal tissue) or systemic (e.g., blood,lymph, etc.). The diagnostic assays of the invention can be invasive ornon-invasive. The diagnostic assays can provide evidence of oxidativerisk (risk of entering the induction phase of ulcerative colitis), ofoxidative stress (in the induction phase), and of oxidative damage(pre-propagation phase).

The subject invention also concerns a method for assessing anindividual's risk of developing ulcerative colitis or determining whatstage of the disease the patient is in. The method comprises assayingfor H₂O₂, glutathione, and exposure time parameters, and thendetermining an individual's oxidative risk (R_(ox)) index based upon thefollowing formula:

$R_{ox} = \frac{( {H_{2}O_{2}\mspace{14mu}{level}} )( {{tissue}\mspace{14mu}{exposure}\mspace{14mu}{time}} )}{( {{glutathione}\mspace{14mu}{level}} )}$whereinH₂O₂ level—Refers to colonic epithelial H₂O₂ level which may bedetermined directly or indirectly.Tissue exposure time—Determined by the colonic epithelial turnover rate.This is the time epithelial cells are exposed to damaging oxygenradicals including H₂O₂.Glutathione level—Refers to the amount of cytoprotective intracellularglutathione available to colonic epithelial cells to neutralize hydrogenperoxide.R_(ox)—A global measure of the oxidative stress level a tissue ororganism is subjected to. The higher the R_(ox) the greater the risk ofdeveloping oxidative medicated tissue damage such as ulcerative colitis.

Assays for determining each of the parameters of the formula aredescribed herein. The R_(ox) value can be used to assess whether anindividual is 1) at risk of entering the induction phase of ulcerativecolitis, 2) is in the induction phase of ulcerative colitis, or 3) is atrisk of entering the propagation phase of ulcerative colitis.

Assays Related to Levels of H₂O Include:

1. Tests that directly or indirectly measure intracellular orextracellular colonic hydrogen peroxide, hydroxyl radical or hydroxideion. Hydrogen peroxide can be assayed in the feces, in rectal biopsytissue, urine, and the blood. Hydrogen peroxide is the final commonpathway for the formation of all hydroxyl (and hydroxide) which are theelements that cause tissue damage. The hydrogen peroxide level is adirect indicator of the potential for oxidative tissue damage. Thus, anycondition that increases H₂O₂ will increase the R_(ox).

2. Tests that directly or indirectly measure the activity of colonicglutathione peroxidase (GPx) enzyme. GPx is the major enzyme thatdegrades hydrogen peroxide and hydrogen peroxide will increase withdecreased GPx activity.

3. Tests that directly or indirectly measure colonic epithelial celllipid membrane peroxidation. Lipid peroxidation in ulcerative colitis iscaused by the effect of hydroxyl radical on the colonic epithelial cell.

4. Tests that directly or indirectly measure colonic epithelial cellproliferation. Hydrogen peroxide induces colonic epithelial cells tomultiply. This is manifested by the presence of Melanosis Coli which isa dark lipid pigment which accumulates in cells that are rapidlydividing. It looks like black spots on the colonic mucosa. Thus, assaysthat measure melanosis coli are contemplated by the invention.

5. Tests that directly or indirectly measure colonic epithelial cellnuclear DNA oxidation (e.g., 8-hydroxy deoxyguanosine (8-OHdG)).Hydrogen peroxide can also diffuse into the nucleus and oxidize the DNA.These oxidation products can be measured and provide an estimate ofoxidative stress.

6. Tests that directly or indirectly measure colonic epithelial cellmitochondrial DNA damage. Hydrogen peroxide is produced in themitochondria and will damage mitochondrial DNA before any otherstructure.

7. Tests that directly or indirectly measure protein carbonyl groupssince hydrogen peroxide can oxidize proteins. Also, tight junctionprotein remnants would be found in feces if oxidized and damaged. Thetight junctions and intracellular enzymes are made of proteins.

8. Tests that directly or indirectly measure colonic epithelial cellsuperoxide levels. Superoxide is the immediate precursor of hydrogenperoxide. If superoxide is elevated then hydrogen peroxide will beelevated.

9. Tests that directly or indirectly measure colonic epithelial cellsuperoxide dismutase activity. Superoxide dismutase is the enzyme thatconverts superoxide to hydrogen peroxide. If its activity is increasedthen hydrogen peroxide will increase.

10. Tests that directly or indirectly measure electron transportactivity. Electron transport activity is responsible for hydrogenperoxide production. If electron transport activity could be measuredthis would be an indirect reflection of hydrogen peroxide production.Electron transport activity is mainly responsible for an individual'sbasal metabolic rate (BMR). When metabolism is increased the electrontransport activity is increased and more hydrogen peroxide is produced.

11. Tests that directly or indirectly measure colonic epithelial cellapoptosis. Hydrogen peroxide causes single cell death called apoptosis.These cells would slough off into the feces. In one embodiment,apoptosis is measured from fecal samples or colonic biopsies.

Assays Related to Time of Exposure:

1. The turnover of colonic epithelial cells is about 3 days. This can beaccelerated by epidermal growth factor. Any condition that decreases EGFor its receptors on the colonic epithelial cells will increase colonicepithelial exposure and R_(ox). Both EGF and its receptors are decreasedin the elderly. There is also an increase in ulcerative colitis inpeople 60 and older. Thus, the measurement of levels of EGF and itsreceptors is a useful diagnostic indicator of R_(ox).

Assays Related to Glutathione Levels Include:

1. Tests that directly or indirectly measure levels of the reduced formof glutathione (GSH) in blood, colonic tissue or feces. When GSH isdecreased, hydrogen peroxide will go un-neutralized and will increase.Only the reduced form of glutathione can neutralize hydrogen peroxide.

2. Tests that directly or indirectly measure the oxidized form ofglutathione in blood, colonic tissue or feces. Oxidized glutathione(GSSG) is produced after hydrogen peroxide is neutralized. If there istoo much hydrogen peroxide to eliminate, GSSG will increase.

3. Tests that directly or indirectly measure glutathione reductaselevels. Glutathione reductase is an enzyme necessary to convert oxidizedglutathione (GSSG) to the reduced form (GSH). If the activity of thisenzyme is decreased, then hydrogen peroxide will increase causing anincreased R_(ox).

4. Tests that directly or indirectly measure NADPH levels. NADPH is thecritical reducing agent that donates electrons to (GSSG) so it can beregenerated back to GSH (reduced glutathione). If NADPH is decreased,then hydrogen peroxide will increase.

5. Tests that directly or indirectly measure G-6-PD (glucose-6-phosphatedehydrogenase), H-6-PD (hexose 6-phosphate dehydrogenase) and/or 6-PGD(6-phosphogluconate dehydrogenase activity. Almost all the NADPH neededto regenerate oxidized glutathione (GSSG) back to the useful reducedform (GSH) necessary to neutralize hydrogen peroxide is produced by onebiochemical pathway, the phosphate pentose pathway (PPP) (formally knownas the hexose monophosphate shunt). Within the PPP two dehydrogenaseenzymes are exclusively responsible for the production of all the NADPHgenerated. These are G-6-PD (glucose-6-phosphate dehydrogenase, EC1.1.1.49) and 6-PGD (6-phosphogluconate dehydrogenase, EC 1.1.1.44).Genetic deficiency of G-6-PD (the ‘G’ form) has been described and willcause hemolysis of red blood cells which lack a normally occurringback-up enzyme known as H-6-PD (hexose-6-phosphate dehydrogenase, the“H” form) present in other cells of the body. Presence of the both the Gand H forms allows the PPP to produce a normal amount of NADPH providingthere is normal activity of the second NADPH producing enzyme, 6-PGD.However, 6-PGD (6-phosphogluconate dehydrogenase) does not have aneffective back up and can be present in a polymorphic form withdecreased activity of up to 50%. This could theoretically decrease totalcolonic epithelial NADPH by as much as 25%. Decreased NADPH will allowhydrogen peroxide to increase elevating the R_(ox) predisposing thecolon to oxidative injury.

6. Tests that directly or indirectly measure butyrate levels. If colonicepithelial butyrate levels are low, then glutathione will be decreased.This will cause hydrogen peroxide to increase.

7. Tests that directly or indirectly measure the levels of glycine,cysteine and glutamic acid. If any of the three amino acids necessaryfor glutathione production (glycine, cysteine and glutamic acid) arelow, then less GSH will be produced and hydrogen peroxide will increase.

Factors associated with increased and decreased risk of inflammatorybowl diseases have been identified. Factors that increase the risk ofdeveloping inflammatory bowl diseases include stress, alcoholconsumption, diet and xenobiotic metabolism. Thus, the subject methodsalso include modification of patient behavior, either alone or incombination with the administration of a therapeutic composition of theinvention.

Positive Risk Factors (Increased Risk of Induction):

Rectum:

The greatest risk factor for ulcerative colitis is the presence of arectum. Within this area of the colon a junction of physiologicalparameters converge to increase the potential for H₂O₂ generation andoxidative tissue damage (induction). Studies have shown that thereducing power (antioxidant capacity) and GSH synthetic rate of the ratintestine decreases distally, reaching its lowest level in distal colonwhen compared to the proximal small intestine (Blau, 1999). The reducingpower of the human gastrointestinal tract likewise decreases fromstomach to colon (Roediger and Babige, 1997). The reducing power of thehuman colon is significantly decreased when compared to the liver withlevels of major anti-oxidant enzymes (catalase, superoxide dismutase andglutathione peroxidase) being 4, 8, and 40% of hepatic valuesrespectively (Grisham et al., 1990). However, rectal epithelial cellshave the same complement of enzymes, i.e., cytochrome oxidase, xanthineoxidase, etc., as the liver for use in metabolism of foreign substancesincluding alcohol and xenobiotics (Roediger and Babige, 1997). Oneby-product of this metabolism is hydrogen peroxide that the rectalepithelium is ill equipped to deal with if produced in excess.Un-neutralized excess H₂O₂ may diffuse to the extracellular space andinduce oxidative damage to the colonic epithelial barrier therebyincreasing its permeability to luminal bacterial antigens. Any substancefound in the body which, through its metabolism, can give rise tohydrogen peroxide in rectal epithelial cells is a positive risk factorfor ulcerative colitis induction.

The bacterial concentration within the colon increases distally from 10³cfu/gram of fecal material in the cecum to 10¹³ cfu/gram in the rectum.The greatly increased concentration of, predominantly, mucosal adherentanaerobic bacteria significantly increases the likelihood of antigenicexposure in the event of a barrier breakdown. Any condition which slowscolonic fecal transport allows bacteria more time to multiply. Thisincrease in intra luminal bacterial concentration is a positive riskfactor for ulcerative colitis induction. High concentrations of bacteriaalso increase the likelihood of horizontal transmission of geneticmaterial including antibiotic resistance and virulence factors.

Fecal material also generates a large amount of oxygen radicals, in partdue to the high concentration of bacteria in addition to the fecalmatrix and other luminal substances including xenobiotics, toxins andbile acids (Owen et al., 2000, Harris et al., 1992). The increasedconcentration of these radicals found in the rectum, a fecal reservoir,can also contribute to colonic barrier dissolution and epithelialoxidative damage. Therefore, any condition which increases colonicbacterial concentration or metabolism will increase the production ofluminal oxygen radicals and is a positive risk factor for ulcerativecolitis induction.

Rectal epithelial cells also possess an electron transport chain whichmay become a source of excess H₂O₂ if subjected to hypoxia and suddenre-oxygenation (Li and Jackson, 2002). During the time the rectum ishousing feces and until they are expelled the rectal wall is subjectedto lateral pressure which can compress delicate sub-mucosal bloodvessels. During this time the rectal epithelial cells are exposed to arelative hypoxia which is suddenly followed by swift reperfusion as thefecal material is expelled. This process of hypoxia and re-oxygenationincreases the activity of the electron transport chain resulting inincreased production of hydrogen peroxide. Any condition that induces orincreases constipation significantly magnifies this effect and is a riskfactor for entering the induction phase of ulcerative colitis.Approximately 10 to 16% of ulcerative colitis patients have fecal stasisand investigators have observed that slow colonic transit may predisposeto ulcerative proctitis (Allison and Vallance, 1991; Black et al.,1987).

Another contributing factor to the production of hydrogen peroxideduring hypoxia and re-oxygenation is the effect of xanthine oxidase(Granger and Parks, 1983; Parks et al., 1984; Parks and Granger, 1986).Xanthine oxidase catalyzes the conversion of hypoxanthine to uric acidand in the process reduces molecular oxygen to hydrogen peroxide. Duringhypoxia xanthine oxidase activity is significantly reduced due tounavailability of oxygen needed as an electron accepting co-factor forthe enzymatic conversion (oxidation) of hypoxanthine to uric acid. Whenoxygen is reintroduced an increased substrate load leads to increasedhypoxanthine metabolism and hydrogen peroxide production. Constipationand straining during defecation, by increasing colonic intraluminalpressure, will enhance the effect of hypoxia/reoxygenation upon thexanthine oxidase enzyme system, increasing its activity and output ofH₂O₂ which increases the risk of entering the induction phase ofulcerative colitis. Allopurinol, an inhibitor of xanthine oxidase, candecrease the production of H₂O₂ by colonic epithelial cells under theseconditions.

Stress:

Stress has several effects on the gastrointestinal tract. Sleepdeprivation stress increases the bacterial translocation through theintestinal epithelium (Everson and Toth, 2000). Stress also activatesintestinal mast cells with subsequent increase in permeability due tomodulation of mucosal barrier function (Soderholm et al., 2002).Psychological and physical stress are reported to cause enterocytephysiology abnormalities, goblet cell dysfunction, increased mucosalbacterial adherence and increased intestinal permeability and secretion(Soderholm and Perdue, 2001).

Stress also increases the amount of circulating biogenic amines(catecholamines), such as serotonin, epinephrine, nor-epinephrine anddopamine (Lechin et al., 1996). Mono-amine oxidase (EC#1.4.3.4), anenzyme present on the outer surface of mitochondria, catalyzes theoxidative deamination of both xenobiotic amines as well as theaforementioned catecholamine stress hormones and in the process reducesmolecular oxygen to hydrogen peroxide (Cadenas and Davies, 2000). Thereaction catalyzed is: RCH₂+H₂O+O₂→RCHO+NH₃+H₂O₂

Therefore, any condition of sustained stress can increase theconcentration of circulating endogenous catecholamines and boostproduction of hydrogen peroxide by rectal epithelial cells. This is apositive risk factor for entering the induction phase of ulcerativecolitis. Likewise, measures aimed at reducing circulating endogenouscatecholamine with reduced H₂O₂ production by colonic epithelial cellsand decrease the risk of rectal epithelial barrier damage. Clonidine, acentral alpha 2 agonist has been used successfully in this regard. Thus,the methods of the present invention also contemplate modification ofpatient behavior and/or physiology to reduce stress on the patientand/or the effects of stress on the patient.

Xenobiotics:

The majority of oxidation reactions of drugs and other xenobiotics arecarried out by Cytochrome enzymes. One study reports 56% of over 300drugs tested are metabolized via the Cytochrome p450 (CYP) family ofoxygenase enzymes present in the endoplasmic reticulum (Bertz andGranneman, 1997). CYP is mostly found in the liver but is also presentin the intestine. A typical CYP catalyzed reaction is:NADPH+H⁺+O₂+RH→NADP⁺+H₂O+R—OH.

This reaction consumes NADPH which is also used in regeneration ofreduced glutathione. Glutathione is the major antioxidant involved inthe neutralization of intracellular hydrogen peroxide. Due to theextensive inter-individual polymorphism observed in the CYP enzymesystem certain individuals may have greater activity then others and canmetabolize xenobiotics at a faster rate consuming more NADPH in theprocess. These individuals are called fast metabolizers. Fastmetabolizers are at a greater risk of entering the induction phase ofulcerative colitis when exposed to certain drugs.

Alcohol:

After ingestion, alcohol is distributed to all cells of the bodyincluding the rectal epithelial cells. Alcohol is enzymaticallyconverted to acetaldehyde by alcohol dehydrogenase. The acetaldehyde isenzymatically converted to acetic acid by aldehyde dehydrogenase. Bothof these cytosolic enzymes utilize NAD⁺ to oxidize their respectivesubstrates and generate NADH that normally serves as an electron donorto the electron transport chain. The increased availability of NADH canactivate the electron transport chain and generate excess hydrogenperoxide (Hoek et al., 2002; Bailey, 1999).

Alcohol can also be metabolized in the endoplasmic reticulum bycytochrome p450 2E1 which depletes NADPH needed for glutathioneregeneration. Alcohol can therefore generate H₂O₂ and decreaseproduction of glutathione needed for neutralization of hydrogenperoxide. High H₂O₂ and depleted glutathione increases the risk ofentering the induction phase of ulcerative colitis.

Alcohol inhibits glutathione peroxidase; a crucial enzyme thatneutralizes H₂O₂, and depletes mitochondrial glutathione (Hoek et al.,2002). Glutathione is not synthesized within mitochondria and must betransported from the cytoplasm into the mitochondria through themitochondrial membranes. Alcohol interferes with the active transport ofglutathione into the mitochondria (Maher, 1997). This leads tomitochondrial depletion of glutathione and H₂O₂ accumulation. Depletedglutathione and increased H₂O₂ levels enhance the risk of entering theinduction phase of ulcerative colitis. Thus, the methods of theinvention contemplate modification of patient intake of alcohol andalcoholic beverages.

Constipation:

Based on surveys of bowel habits constipation occurs in up to 20% of thepopulation (Drossman et al., 1999; Everhart et al., 1987). Constipationis reported to occur in 27% of patients with ulcerative colitis (Rao,1988). Constipation is a common but often an overlooked condition thatcan have a serious exacerbating impact on almost any disease especiallyulcerative colitis. At the 13th International Congress ofGastroenterology in Rome, Italy in 1988 a group of physicians definedcriteria to more accurately diagnose several different functional boweldisorders including constipation. Known as the “Rome Criteria,” this setof guidelines outlines symptoms and applies parameters such as frequencyand duration in order to make possible a more accurate and standardizeddiagnosis of constipation. In 1999, a second set of updated guidelines,Rome II, was published (Drossman et al., 2000). The Rome II definitionof functional constipation consists of the following parameters:

-   -   At least 12 weeks, which need not be consecutive, in the        preceding 12 months of two or more of:    -   1. Straining in over 25% of defecations;    -   2. Lumpy or hard stools in over 25% of defecations;    -   3. Sensation of incomplete evacuation in over 25% of        defecations;    -   4. Sensation of anorectal obstruction or blockade in over 25% of        defecations;    -   5. Manual maneuvers to facilitate over then 25% of defecations        (e.g. digital evacuation, support of pelvic floor) and/or    -   6. Less than 3 defecations per week.    -   Loose stools are not present, and there are insufficient        criteria for IBS.

As stated above, constipation is a significant exacerbating factor forulcerative colitis. Twenty seven percent of individuals with ulcerativecolitis have coexisting constipation. Constipation can contribute to thedevelopment of the induction phase of ulcerative colitis in severalways. Increased colonic transit time favors bacterial overgrowth whichincreases the antigenic stimulus once the colonic epithelial barrier isdisrupted. Constipation also allows for greater contact time betweenfecal generated oxygen radicals and the colonic mucosa which may depletethe already low antioxidant stores of the epithelial lining.

Large accumulations of un-expelled rectal fecal material may increaselateral pressure on the rectal mucosa and collapse delicate sub mucosalcapillary beds predisposing to a reoccurring perfusion-reperfusionoxidative insult to the colonic epithelium. Constipation willsignificantly decrease the amount of butyrate reaching the rectalepithelium due to proximal colonic absorption and further exacerbateoxidative injury due to glutathione depletion. Thus, the methods of theinvention contemplate treatments and/or modification of patient diet torelieve and/or minimize constipation and to provide for regular bowelmovement.

Diet:

Butyrate:

Dietary factors can contribute to induction of ulcerative colitis. Thepreferred energy source for colonic epithelial cells is a four chainfatty acid known as butyrate. It originates from two dietary sources.Most butyrate is derived from colonic bacterial fermentation ofunabsorbed dietary fiber and is the single largest metabolite of dietaryfiber. The second significant source dietary source is from butter whichcontains 3% butyrate. Butyrate is a short chain fatty acid (SCFA) and isproduced along with two other SCFAs (acetate and propionate) as a resultof bacterial fermentation of dietary fiber (Topping and Clifton, 2001).Ninety-five percent of SCFAs are produced and absorbed in the colon.Fermentation and SCFA production are high in the proximal large bowel.During passage of the fecal stream, fermentation declines secondary todepletion of available substrate. By the time the fecal mass reaches therectum less than 5% of bacterially derived SCFA are available forcolonocyte uptake. Proximal colonic absorption is responsible for thissignificant decline.

SCFAs are the metabolized rapidly by colonocytes and are the majorrespiratory fuels supplying 70% of colonocytes energy needs (Topping andClifton, 2001). Colonocytes prefer to use butyrate even when competingsubstrates such as glucose, glutamine and other SCFAs are present.

The distal colon and rectum are the regions of the large intestine withthe most limited supply and slowest absorption of butyrate and is thesite of most pathology including ulcerative colitis. When the amount ofbutyrate is insufficient to meet the energy needs of colonic epithelialcells an alternate source of energy is utilized and the colonicepithelial cells will switch to glucose and the amino acid glutamine.

Glutamine is synthesized from glutamic acid, its immediate precursor.Glutamic acid is also used to synthesize glutathione which is needed toneutralize hydrogen peroxide. If too much glutamic acid is diverted tothe formation of glutamine as an energy source then less is availablefor colonic epithelial glutathione synthesis. This can result in adiminished intracellular glutathione concentration with a correspondingincrease in hydrogen peroxide. Therefore, the lack of dietary fiber is apositive risk factor for entering the induction phase of ulcerativecolitis.

On a wider scale, 90% of butyrate and other SCFAs are metabolized viabeta-oxidation within mitochondria of colonocytes (10% withinperoxisomes). Conditions that interfere with beta-oxidation will forcethe utilization of an alternate energy source such as glutamine.Beta-oxidation is decreased when sufficient substrate (i.e., butyrate)is unavailable to the rectal epithelial cells. Inadequate rectal luminalbutyrate can occur with inadequate ingestion of fermentable dietaryfiber or increased colonic transit time (constipation). Butyrate cannotbe detected in feces at whole gut transit time exceeding 50 hours(Topping and Clifton, 2001).

Roediger and Nance report the induction of ulcerative colitis in ratsafter rectal instillation of a specific inhibitor of beta-oxidation(Roedinger, 1986). The authors conclude that “a suitable inhibitor ofbeta-oxidation would have unimpeded entry into mitochondria of colonicepithelial cells.” Hydrogen peroxide is permeable through biomembranesincluding the cell membrane and both the inner and outer mitochondrialmembranes. Hydrogen peroxide has been shown to inhibit thebeta-oxidation enzyme system of enzymes (Gulati et al., 1993). H₂O₂produced as a consequence of neutrophil activation within colonic mucosaduring active ulcerative colitis is therefore capable of diffusing backinto colonic epithelial cells resulting in oxidative damage tointracellular proteins including enzymes of beta-oxidation. This wouldexplain the abnormalities of butyrate metabolism during activeulcerative colitis which resolve during remission. Thus, the methods ofthe invention also contemplate modification of patient diet to providefor sufficient glutathione in colonic epithelial cells including, forexample, providing for sufficient dietary fiber in a patient beingtreated or at risk for developing an inflammatory bowel disorder.

Fat:

High intakes of mono and poly unsaturated fat may enhance the risk ofdeveloping ulcerative colitis. The concentrations of lipid peroxidesfound in common foods cooked in oils or fats (hamburger, French fries)can produce mucosal oxidative stress and redox imbalance when in contactwith intestinal mucosa (Aw, 1999). Rats fed high fat diets had sevenfold greater intestinal production of superoxide when compared tocontrols. Intestinal lipid peroxidation and hydroxyl radical wasincreased 3.5 fold. Intestinal mucosal DNA fragmentation was increased2.4 times (Bagchi et al., 1998). Oxidative DNA damage suggests thatthere is a high cytoplasmic oxidative environment. The presence of highfat intake is a positive risk factor for entering the induction phase ofulcerative colitis. Thus, the methods of the invention also contemplatemodification of patient diet to decrease intake of fat.

Spices:

Spicy food was found to produce similar oxidative stress on intestinalmucosa as a high fat diet. Intestinal mucosal hydroxyl radicalproduction in rats fed spicy foods was found to be 4.8 times normal,even greater than rats fed a high fat diet (Bagchi et al., 1998). Spicyfood intake should be considered a positive risk factor for entering theinduction phase of ulcerative colitis.

Meat:

A diet high in red meat (beef) has been reported to increase theintestinal Bacteroides population (Maier et al., 1974). This effectpersisted for several weeks after terminating the high beef diet andwhile on a normal diet regimen. Bacteroides species have been implicatedas the inciting antigenic agent in ulcerative colitis (above).

Vitamins and Minerals:

Folic Acid:

Methylenetetrahydrofolate reductase (MTHFR) (EC 1.5.1.20) is one of themain regulatory enzymes of homocysteine metabolism and plays a majorrole in the metabolism of folates (Friedman et al., 1999; Goyette etal., 1998). MTHFR is a cytoplasmic enzyme that catalyzes theNADPH-linked reduction of methylene-tetrahydrofolate tomethyl-tetrahydrofolate. Methyl-tetrahydrofolate serves as the methyldonor for the methylation of homocysteine. Mutations in the gene codingfor MTHFR have been found. This genetic mutation results in a cytosineto thymine transition at base position 677 which converts alanine tovaline at amino acid position 222 (A222V). The polymorphic MTHFR enzyme(C677T) has significantly less activity than normal (35-50% of normal)resulting in an elevation of serum homocysteine levels (Goyette et al.,1998).

Mahmud et al. report that 17.5% of individuals with ulcerative colitisare homozygous for the C677T variant of the MTHFR gene versus 7.3% ofcontrols (Mahmud et al., 1999).

Elevated homocysteine will increase hydrogen peroxide production byseveral mechanisms. H₂O₂ is generated during the oxidation ofhomocysteine to homocystine (Friedman et al., 1999; Upchurch, et al.,1997). Homocysteine also increases superoxide dismutase (SOD) levels(Wilcken et al., 2000). SOD catalyzes the conversion of superoxide anionto hydrogen peroxide. Increased activity of this enzyme will result ingreater H₂O₂ generation. Homocysteine has been reported to inhibitglutathione peroxidase (GPx) activity (Upchurch et al., 1997) by 10 fold(Outinen et al., 1999). GPx is an essential enzyme system whichneutralizes intracellular H₂O₂. Inactivation of GPx will increase H₂O₂levels and inhibition of GPx was shown to occur at physiologic (9micromol/L) concentrations of free homocysteine (Chen, 2000).

Biogeographically, the worldwide distribution of the C677T polymorphicvariant also points to a role for the deleterious effects of thismodified enzyme in the development of ulcerative colitis. The T allelewas found to have a frequency of 40% in North America but was very lowin Africa and Asia (The Metabolic and Molecular Basis of InheritedDisease, 2001, 8th ed., Chapter 155, Vol. 3, pp. 3897-3993). Thisparallels the incidence of ulcerative colitis which is more common inNorth America and less common in Asia and Africa (Whelan, 1990;Farrokhar et al., 2001).

The ethnic distribution of C677T also suggests a role in ulcerativecolitis. Rady et al. found that the rate of C677T among Ashkenazi Jewishalleles was 47.7% compared to 28.7% among the non-Jewish population(Rady et al., 1999). Ulcerative colitis is also reported to be morecommon in the Jewish population (Roth et al., 1989; Karlinger, 2000).

In other words, this variant MTHFR enzyme can increase colonicepithelial intracellular hydrogen peroxide by increasing SOD levels,inactivating glutathione peroxidase, inhibition of glutathione synthesisand increasing production of homocysteine which generates H₂O₂ duringits oxidation. The physiological effects of the C677T variant of MTHFRmay contribute enough oxidative stress in the form of additional H₂O₂ totip the balance towards entering the induction process of ulcerativecolitis.

The oxidative phenotype associated with C677T may have enough penetranceto predispose certain ethic groups and geographical populations tomanifest a higher incidence of ulcerative colitis. Therefore, thepresence of this polymorphic MTHFR should be considered a positive riskfactor for entering the induction phase of ulcerative colitis.

Additionally, the lack of normal folate metabolism may decreaseavailability of the essential amino acid methionine which is a precursorof the amino acid cysteine. Cysteine is one of the three peptides neededto synthesize glutathione. Glycine, another component amino acid ofglutathione, also requires folate for its synthesis. Folate deficiencyor inhibition of folate metabolism may therefore directly interfere withadequate production of glutathione and contribute to increasedintracellular oxidative stress via increased hydrogen peroxide levels.

Low serum level of multiple vitamins, including folate has been reportedin association with ulcerative colitis (Fernandez-Banares et al., 1989;Elsborg and Larsen, 1979; Koutroubakis et al., 2000). This has beenattributed to the effects of inadequate diet, intestinal malabsorptionor drug induced (Elsborg and Larsen, 1979). Co-existing folatedeficiency would tend to exacerbate the oxidative effects of MTHFRpolymorphism as indicated above. In monkeys, experimentally inducedfolate deficiency leads to colonic ulcerations (Duncan, 1964).

Vitamin B-6:

High intakes of pyridoxine (vitamin B-6) have been reported to enhancethe risk of ulcerative colitis (Geerling et al., 2000). Vitamin B-6 isoxidized in the body by Pyridoxine 4-oxidase to pyridoxal via thereaction: Pyridoxine+O₂→pyridoxal+H₂O₂. This mainly occurs in the liverbut can occur in other places such as the gastrointestinal tract. Atoxic by-product of this reaction is hydrogen peroxide. Therefore,excessive pyridoxine is a positive risk factor for entering theinitiation phase of ulcerative colitis.

Iron and Copper:

A single electron reduction is the only type of reaction which leads tothe formation of hydroxyl radical from hydrogen peroxide. The electrondonating agent may be an electron donating radical such as superoxide(O₂ ⁻.) or a biologically significant redox transition metal such asiron or copper (Eberhardt, 2001). These one electron reduction reactionsof H₂O₂ and other peroxides represent the most important radical formingreactions in biological systems and are responsible for most hydroxylgeneration in human cells.Fe⁺²+H₂O₂→Fe⁺³+HO⁻+HO.  Fenton reactionO₂ ⁻.+H₂O₂→O₂+HO⁻+HO.  Haber-Weiss reaction

Iron can act as an intermediate and facilitate the transfer of a singleelectron from superoxide to hydrogen peroxide. The presence of iron willaccelerate the generation of hydroxyl radical from hydrogen peroxide viathe iron catalyzed Haber-Weiss reaction (Eberhardt, 2001; Graf et al.,1984).O₂ ⁻.+Fe⁺³→O₂+Fe⁺²  Ferric Iron (Fe⁺³) catalyzedFe⁺²+H₂O₂→Fe⁺³+HO⁻+HO.  Haber-Weiss reaction.

Within cells, iron is coupled to biological macromolecules such asproteins and DNA. The formation of hydroxyl radical will therefore takeplace in close contact with these molecules. Since the hydroxyl radicalis highly reactive, the damage caused by this very powerful oxidizingagent occurs at the site of formation (Eberhardt, 2001). This rapid sitespecific target oxidation prevents radical scavengers from interferingwith this reaction and greatly magnifies the damaging effects of theH₂O₂/HO. system. Even in the absence of readily available reactivesurface iron atoms, superoxide can induce the release of iron fromstorage proteins such as ferritin and enzymes. This released iron isthen able to generate hydroxyl by reacting with hydrogen peroxide (Keyerand Imlay, 1996; Liochev and Fridovich, 1999). Thus, within biologicalsystems, superoxide is able to provide a steady supply of its own ironcatalyst needed to perpetuate the production of hydroxyl from hydrogenperoxide. Conversely, iron overload can damage tissues even whensuperoxide concentrations are minimal, suggesting that other reducingagents besides O₂ ⁻. can supply the electrons necessary to free ironwhen it redox-cycles with H₂O₂ (Keyer and Imlay, 1996).

In accordance with these observations, iron supplementation has beenreported to damage the GI tract via the formation of oxygen radicals andto worsen experimentally induced colitis in laboratory animals(Srigiridhar et al., 2001; Reifen et al., 2000). Human ulcerativecolitis has also been reported as a consequence of oral ferrous sulfatetreatment for anemia (Kawai et al., 1992). Conversely, iron chelationhas been reported to reduce the production of reactive oxygen species inpatients with ulcerative colitis (Millar et al., 2000).

Excessive iron intake in the form of supplements or red meats (i.e.,beef), therefore, may enhance the production of (GI barrier) damaginghydroxyl radical and facilitate the transition from the induction phaseto the propagation phase of ulcerative colitis. Excessive iron istherefore a risk factor for entering the propagation phase of ulcerativecolitis.

Copper is reportedly present in higher than normal amounts in active andquiescent human ulcerative colitis (Dalekos et al., 1998; Ringstad etal., 1993) and may be a contributing factor in the formation of hydroxylradical. Human ulcerative colitis has been reported in association withand subsequent to the development of Wilson's disease in whichabnormally high copper levels are present in body tissues (Torisu,2002). As with iron, excessive copper can accelerate the reduction ofH₂O₂ and increase the generation of hydroxyl radical. Excessive coppercan therefore be considered a risk factor entering the propagation phaseof ulcerative colitis.

Vitamin C:

Vitamin C (ascorbic acid) is an antioxidant that reacts with oxygenradicals. However, serial oxidations of the ascorbyl radical cangenerate hydrogen peroxide that gives rise to the hydroxyl radical inthe presence of iron or copper (Eberhardt, 2001). Combined ascorbic acidand mineral supplements can be a positive risk factor for entering theinduction phase of ulcerative colitis. Ascorbate can also be oxidized byL-ascorbate oxidase which generates hydrogen peroxide as a by product.Excessive intake of vitamin C of itself may be a contributing riskfactor induction in ulcerative colitis.

Vitamin B-1:

Thiamine (Vitamin B-1) can be metabolized by Thiamine oxidase whichgenerates H₂O₂. Excess thiamine can be a contributing risk factor forentering the induction phase of ulcerative colitis.

Artificial Sweeteners:

Artificial sweeteners such as Sorbitol and Xylitol are oxidized byXylitol oxidase which generates hydrogen peroxide. If consumed in largeamounts can be a risk factor for induction in ulcerative colitis.

Aspartame is an artificial sweetener used extensively in many productsincluding soft drinks and baking goods. It is commonly seen in a smallblue packet along with sugar and saccharine in many restaurants.Aspartame is a dipeptide composed of two amino acids, L-phenylalanine asthe methyl ester (Phe) and L-aspartic acid (Asp). About 10 percent byweight of aspartame is released as methanol. Methanol can be metabolizedby methanol oxidase to formaldehyde and H₂O₂. Aspartic acid ismetabolized by aspartic oxidase with the release of H₂O₂. Phenylalanineis metabolized by amino acid oxidase with the subsequent release ofH₂O₂. The amount of oxidative stress caused by aspartame depends on theamount ingested. Since this product is so ubiquitous its contribution tocolonic epithelial hydrogen peroxide may exceed that from generalprotein amino acid intake and may be a risk factor for induction insusceptible individuals.

Monosodium Glutamate:

Monosodium glutamate (MSG) is the sodium salt of the amino acid glutamicacid.

It is used in high concentrations as a flavor enhancer. Glutamic acid ismetabolized by glutamate oxidase and hydrogen peroxide is released as aby-product of this reaction. The oxidative load of MSG will depend onthe amount consumed. Susceptible individuals that consume a large amountof MSG may be at risk for entering the induction phase of ulcerativecolitis.

Thus, the methods of the invention also contemplate modification of theintake of and levels of vitamins, artificial sweeteners, and foodadditives in a patient.

Negative Risk Factors (Decrease Risk of Induction):

Certain factors have been found to decrease the risk of developingulcerative colitis. Chief among them is the association of cigarettesmoking with a decreased incidence of developing ulcerative colitis(Abraham et al., 2003; Odes, 2001). Some investigators have labeledulcerative colitis a disease of non-smokers (Madretsma, 1996). The riskof developing ulcerative colitis is greatest among individuals who haverecently quit smoking followed by non-smokers (Farrell and Peppercorn,2002).

Tar products in tobacco smoke have been shown to inhibit the electrontransport chain. Studies quantifying the effect of cigarette tar onmitochondrial electron transport activity report an 82% inhibition rateon whole chain respiration (Pryor et al., 1992). In this study nicotinehad no effect on electron transport activity. ETC inhibition will reducemitochondrial hydrogen peroxide generation. Cigarette smokers have alsobeen reported to have reduced monoamine oxidase activity (Fowler et al.,2003). MAO, located on the mitochondrial outer membrane, is the mainenzyme responsible for endogenous catecholamine metabolism. Inhibitionof this enzyme decreases enzymatic oxidation of catecholamines and thegeneration of hydrogen peroxide which is a by-product of this reaction.Extracts of cigarette tar are reported to inhibit a number of P450enzyme systems (Van Vleet et al., 2001). Hydrogen peroxide is aby-product of P450 metabolism and inhibition of these enzymes can lowerH₂O₂ generation.

Cigarette smoke extract has also been reported to inhibit the productionof cytokines, including TNF-alpha, by greater than 90%. (Ouyang et al.,2000). Cytokines are an integral component of the inflammatory responsein ulcerative colitis and their reduction may confer protection.

Thus, smoking may confer protection by inhibiting H₂O₂ production andpreventing the induction phase of ulcerative colitis. However, duringthe time the ETC is inhibited by smoking, reducing equivalents, thesubstrate for H₂O₂ production, have been accumulating withinmitochondria of all cells of the body including colonic epithelialcells. Inhibition of the electron transport chain also causesup-regulation of ETC associated enzymes in an attempt at overcoming theblockade. During the time of ETC inhibition glutathione production mayalso be down regulated since the cell does not have use for high levelsof antioxidants when H₂O₂ production is low.

When an individual suddenly stops smoking this inhibition is abruptlyremoved resulting in greater ETC activity fueled by increased substrateand enzymatic activity. This also results in extra hydrogen peroxidebeing produced which, in susceptible individuals, may overwhelm theavailable glutathione within the colonic epithelial cells and increasethe risk of induction.

Based on ETC inhibition, the risk of induction would be expected to belowest in active smokers. Smokers that have recently quit smoking wouldbe at highest risk since they are producing the most H₂O₂. Non-smokerswould have the lowest risk. This mirrors the statistical risk of smokingas it relates to ulcerative colitis.

Smoking cessation during active ulcerative colitis would therefore beexpected to exacerbate the condition. An increase in severity ofulcerative colitis has been reported in patients who stopped smokingduring active ulcerative colitis (Beaugerie et al., 2001).

Nicotine is a parasympathomimetic and exerts its effect on the GI tractlargely by stimulation of the parasympathetic ganglia. Its effect is togenerally increase tone and contractility (National Academy of Sciences,2001). The nicotine in cigarette smoke enhances intestinal peristalticactivity which tends to increase the fecal stream and prevent fecalstasis which can increase colonic bacterial and epithelial oxidantstress loads. Abrupt cessation of this adjuvant to colonic peristalticactivity would tend to favor stasis of colonic contents and promoteconstipation which is a risk factor for the development of ulcerativecolitis (see constipation above). Thus, the methods of the inventioncontemplate regulated, progressive cessation of smoking in a patient.

Co-Morbid Conditions:

Ulcerative colitis has been associated with hyperthyroidism. In one casereport two patients developed ulcerative colitis while being followedfor hyperthyroidism (Modebe, 1986). One of these patients had a secondexacerbation of ulcerative colitis which could not be controlled untilit was discovered that she was thyrotoxic. The ulcerative colitis onlyresponded to therapy after controlling the hyperthyroidism.

Another study compared the frequency of thyroid disease between 300patients with documented ulcerative colitis and 600-age and sex matchednormal controls (Jarnerot et al., 1975). A history of thyrotoxicosis wasobtained in 3.7% of the ulcerative colitis patients compared with 0.8%of controls. Seventy percent of the ulcerative colitis patientsdeveloped hyperthyroidism prior to developing ulcerative colitis.Although ulcerative colitis patients can subsequently develophyperthyroidism it is unlikely that a subclinical hyperthyroid statewill be uncovered before the patient seeks treatment for ulcerativecolitis and therefore the number of ulcerative colitis patients withpreceding hyperthyroidism may be underestimated.

The colon and the thyroid gland have dissimilar embryological originsarguing against a common cross-reactive antigenic stimulus for thedevelopment of an auto-immune pathogenesis as an etiology in bothdiseases.

Thyroid hormone is known to raise the metabolic rate. It accomplishesthis by increasing the activity of the mitochondrial electron transportchain (Venditti et al., 2003; Goglia et al., 2002; Venditti et al.,1997). Under the influence of excess thyroid hormone greater amounts ofhydrogen peroxide are generated and released by acceleratedmitochondrial ETC activity (Venditti et al., 2003).

Thus, increased ETC activity results in higher intracellular H₂O₂production in all cells of the body including colonic tissue. Underappropriate conditions this can result in colonic epithelium enteringthe induction phase of ulcerative colitis when excess un-neutralizedH₂O₂ diffuses out of the colonic epithelial cell.

Conversely, chemically induced hypothyroidism is reported tosignificantly attenuate animal models of experimental colitis (Isman etal., 2003; Oren et al., 1997). This suggests that H₂O₂ constitutivelyproduced in the euthyroid state presents a major oxidant load to thecell and results in the consumption of significant amounts ofintracellular glutathione.

Thus, the methods of the invention contemplate the use of compositionsand methods to control thyroid hormone levels in a patient.

Genetic Factors:

Ulcerative colitis has an approximate 10% concordancy rate betweenmono-zygotic twins (Farrell and Peppercorn, 2002). This suggests thatenvironmental factors are diverse and very influential in thedevelopment of the disease. The lack of an identifiable genotypesuggests that genetic factors are multiple (not singular) and withinsufficient penetrance to elicit the disease on their own.

In other words, environmental exposure of synergistic factors each ofwhich is beneath the threshold to elicit a recognizable pathogenicresponse on their own may be acting in concert with several geneticfactors of weak penetrance which overlap in only 10 percent ofmonozygotic twins in order for ulcerative colitis to occur.

Genetic research performed by Cho et al. uncovered the existence of apathophysiologically crucial IBD susceptibility gene (1p36) located onthe small arm of human chromosome 1 (Cho et al., 2000; Cho et al.,1998). This genetic locus codes for two enzymes that are able toinfluence the intracellular redox environment at a fundamental level.

The first enzyme, Methylenetetrahydrofolate Reductase (MTHFR, EC1.5.1.20), has several polymorphic variant phenotypes which interferewith normal folate metabolism (above). It is located at 1p36.3. It ispresent in about twenty percent of individuals with ulcerative colitis.This polymorphic enzyme causes elevation in serum homocysteine and adecrease in methionine and glycine. This epistatic effect can inhibitglutathione peroxidase function and interfere with glutathionesynthesis, both of which are essential for detoxification of hydrogenperoxide. This results in higher than normal intracellular H₂O₂ andincreased risk of ulcerative colitis induction (above). Persons withmutant or defective MTHFR might be treated prophylactically with folateand trimethylglycine.

A second enzyme located at this locus (1p36.3) is 6-Phosphogluconatedehydrogenase (PGD) (EC 1.1.1.44). PGD is the second dehydrogenaseenzyme in the pentose phosphate pathway (hexose monophosphate shunt)which is responsible for production of NADPH. PGD catalyzes theconversion of 6-phosophogluconate to ribulose-5-phosphate generating twomolecules of NADPH. NADPH is crucial for the reduction of glutathionedisulfide (GSSG) back to reduced glutathione (GSH) in order toneutralize the continuous production of hydrogen peroxide beinggenerated within the cell. The pentose phosphate pathway is the majorsource of reducing equivalent (NADPH) that allows H₂O₂ generatingsystems, such as the ATP producing electron transport chain and mostoxidase enzymes, to function. Without NADPH to regenerate reducedglutathione, intracellular enzymes would suffer irreversible oxidativedamage from excess hydrogen peroxide and cellular function would ceasewithin minutes as apoptosis is triggered.

PGD exists in several polymorphic forms with decreased activity rangingfrom 22 to 79% of normal (Davidson, 1967; Parr, 1966; Parr, 1967; Dernet al., 1966; Nelson, 1982). Decreased levels of glutathione have beenreported as a result of a PGD polymorphic enzyme (Capari, 2001). Thissuggests that normal activity of both NADPH producing enzymes in thepentose phosphate pathway is necessary for normal glutathione levels.PGD activity can also be lowered by exogenous factors such asantibiotics, dietary fat and the ageing process (Cifici, 2002;Tomlinson, 1998; Gordillo, 1991).

Polymorphic variants of PGD can decrease the amount of NADPH beinggenerated. Under the appropriate conditions, individuals with thisgenetic polymorphism may then be at greater risk for H₂O₂ accumulationwithin the cell and higher risk for ulcerative colitis induction.Compromised PGD activity may, therefore, contribute to the ulcerativecolitis induction process in predisposed individuals.

Cytochrome p450 oxygenase is a part of a family of about 50 geneticallydistinct enzyme systems that metabolize xenobiotics. This enzymeutilizes NADPH as an electron donor in order to metabolize drugs andtoxins that enter the body. As mentioned above NADPH is also needed toregenerate reduced glutathione. Therefore fast metabolizers of anyspecific drug will consume more NADPH when exposed to that drug. Thediversion of NADPH from Glutathione regeneration to drug detoxificationcan increase the intracellular concentration of hydrogen peroxide andlikewise the risk of ulcerative colitis induction. P450 enzymes alsogenerate H₂O₂ during metabolism of xenobiotics further increasing theoxidative load.

Studies of normal appearing colonic mucosa have reported significantinter-individual variation of enzymes involved in glutathione synthesisand metabolism (Batist et al., 1988). Variation between individuals wasconsiderable at 8 fold for glutathione-S-transferase, 10 fold forglutathione peroxidase, 16 fold for glutathione levels, 14 fold forgamma-glutamyl-transpeptidase and 5 fold for gamma-glutamylcysteinesynthetase. These large enzyme variations directly or indirectly affectintracellular glutathione concentrations which itself shows a 16 foldvariation between individuals.

Ethnic variation and enzymopathies has also been reported forphosphogluconate dehydrogenase (EC: 1.1.1.44), glutathione peroxidase(EC 1.11.1.19), glutathione reductase (EC 1.6.4.2),gamma-glutamylcysteine synthetase (EC 6.3.2.2), and glutathionesynthetase (EC 6.3.2.3) (Scriver et al., 2001; Larsson and Anderson,2001; Buetler and Matsumoto, 1975).

Genetic factors affecting colonic glutathione concentrations arenumerous. Factors affecting the synthesis, reductive regeneration, orphase II bio-conjugation of glutathione can increase intracellular H₂O₂.Individually no single factor appears to be able to alter the redoxbalance to the point of excessive intracellular H₂O₂ accumulation.However, in the face of a confluence of these genetic factors and theappropriate environmental conditions the colonic epithelium's ability toneutralize intracellularly generated hydrogen peroxide may becompromised and the tissue can enter the induction phase of ulcerativecolitis.

Thus, the methods of the invention also contemplate genetic screening ofan individual and modification, as necessary, of lifestyle and/orinstitution of treatment regimens based on results of the screening.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing theinvention.

These examples should not be construed as limiting. All percentages areby weight and all solvent mixture proportions are by volume unlessotherwise noted.

EXAMPLE 1 Treatment of Patient During Induction Phase

It has been discovered that ulcerative colitis, like many other diseasescan be prevented if it is recognized during the induction phase.Recognition of the induction process is difficult since there are littleor no symptoms or signs pointing to the colon as the source of theproblem and the colon is histologically and macroscopically normal.Recognition at this stage requires a high index of suspicion coupledwith some knowledge of the extra intestinal manifestations, xenobioticassociations, family and genetic history, ulcerative colitisepidemiological data and life style history.

A p-anca antibody at this time may be positive if the colonic epithelialbarrier has been rendered sufficiently permeable to allow prolongedcontact between the immune system and bacterial antigens in the colon.The p-anca antibody has been shown to be directed against a surfaceantigen of B. Vulgatus and is an indication of colonic barrier breachwith subsequent immune activation. Evidence of increased colonicepithelial turnover may be found in fecal samples since H₂O₂ can induceepithelial proliferation. If a colonoscopy should be performedadditional evidence of epithelial cell proliferation may be seen such asmelanosis coli (Pardi et al., 1998). Immunological staining of colonicbiopsies may reveal altered tight junction proteins such as cadherin andbasement membrane abnormalities. In vivo conductance studies, if thiswere possible, would show increased permeability in macroscopicallynormal colonic tissue.

In patients that are determined to be in the induction phase, measurescan be undertaken to implement lifestyle changes in order to reduce theoxidative stress on the colon. All xenobiotics and alcohol should beterminated. Smoking should be discontinued gradually rather than via acomplete cessation, i.e., the patient should avoid going “cold turkey”when trying to stop smoking. Constipation should be corrected. Fast foodshould be eliminated and a diet high in antioxidants (vegetables andfruit), fiber, and good quality protein should be instituted. Stressreduction should be instituted with counseling if necessary.

EXAMPLE 2 Treatment of Patient During Propagation Phase

Currently, individuals are almost never recognized during the inductionphase and only seek medical help because of rectal bleeding when thepropagation phase has already developed. A colonic neutrophilicinflammatory reaction into the colonic mucosa cannot be reversed withthe same measures used during the induction phase, although it isprudent to institute them in order to prevent re-induction afterreversal of the inflammatory reaction has been accomplished.

Treatment of colonic inflammation during the propagation phase comprisesone or more of the following:

1. Neutralization of colonic hydrogen peroxide.

2. Reduction of neutrophilic stimulation by colonic bacteria.

3. Termination of colonic epithelial cell lipid peroxidation.

4. Reduction of colonic mucosal permeability.

Neutralization of hydrogen peroxide is critical in order to terminatecontinued tissue damage. This can be accomplished, for example, withrectal instillation of sodium thiosulfate that will neutralize hydrogenperoxide to water and non-reactive sulfate products.

The stimulatory effect of colonic bacteria, mainly anaerobicBacteroides, on neutrophils can be mitigated with bismuth subgallatewhich prevents bacterial adherence to the colonic epithelium and isbactericidal.

Termination of colonic epithelial lipid peroxidation can be achievedwith d-alpha tocopherol (vitamin E) as the acetate or the succinate.This also adds viscosity to the solution which creates a sterichindrance to prevent cytokines and radicals from interacting with theirtarget tissue.

Finally, cromolyn sodium can block colonic mast cells and decreasecolonic permeability to luminal antigens.

This therapy can be administered as a retention enema once daily.

Oral therapy with Clonidine to reduce the oxidative effects ofendogenous catecholamines secondary to stress can also be instituted.Pentoxyfylline has anti-inflammatory activity and may function as apurinergic agonist via an adenosine receptor on the surface of theinfiltrating neutrophil which can inhibit NADPH oxidase and apoptosis.This oral therapy can be continued, along with lifestyle changes, asmaintenance therapy to prevent re-initiation and relapse.

EXAMPLE 3

Patient is a 44 year old female who has had ulcerative colitis since1994. It started with bleeding. She actually had bleeding and crampingabout every six months. Each episode abated spontaneously withouttherapy. They began to increase in intensity until 2003.

In October 2003 patient had a colonoscopy and for the first time, anofficial diagnosis of ulcerative colitis was made. She was started onASACOL (Medeva Pharma Schweiz A G, Switzerland) but still hadrecurrences; however, the recurrences were shorter. The recurrencesoccurred every 3-5 months. Some of the episodes were severe but did notrespond to hydrocortisone enemas. Subsequently, she had a very severeepisode that did not respond to ASACOL or the enemas. She was treatedwith prednisone and improved for a few months but then it recurred andwas more severe than ever.

Flexible sigmoidoscopy showed moderately active diseases to exactly 20cm (see FIGS. 1A-1E). Above that, the mucosa was normal and stool wasnormal. Biopsies and pictures were taken. Biopsy specimens showeddiffuse colitis with distortion of crypt architecture, mononuclear cellexpansion of the lamina propria, basal plasmacytosis and mucosalulceration, consistent with chronic ulcerative colitis, severe activity,with epithelial changes negative for dysplasia. CMV was not identified.

Following sigmoidoscopy, patient was started on a once daily enematreatment with an enema formulation of the present inventioncomprising: 1) 40 cc of Rowasa (2.6 gms) (Mesalamine) Rectal Suspension;2) 5 cc sodium cromolyn (100 mg/5 cc oral concentrate); 3) 15 cc of 1Molar sodium butyrate (15 milimoles or 1.5 gms); and 4) 1 cc sodiumbudesonide (5 mg/cc). The patient also was started on oral R-dihydrolipoic acid 300 mg given twice daily.

After completion of 7 days of treatment, patient claims to be 85%improved having noticed improvement beginning about the fourth day. Sheis having no more cramping, no mucus, and no blood. Her stools areforming. Patient will continue for another week on treatment and thenwill be examined by sigmoidoscopy.

Flexible sigmoidoscopy of patient showed an absolutely normal lookingmucus membrane (see FIGS. 2A-2C). Biopsies were taken. Biopsy specimensshowed distortion of crypt architecture and minor mononuclear cellexpansion of the lamina propria consistent with quiescent chroniculcerative colitis. Active inflammation was not identified. Theepithelial changes were negative for dysplasia.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

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I claim:
 1. A method for the treatment of an inflammatory bowel disorderin a person or animal, said method comprising administering an effectiveamount of: a) a composition formulated for rectal administration as anenema or suppository in a person or animal, said composition comprisingfrom about 500 mg to about 5000 mg of 5-ASA (Acetyl Salicylic Acid),from about 1 mg to about 10 mg of budesonide, from about 10 mg to about1000 mg of cromolyn sodium and from about 5 millimoles to about 50millimoles of sodium butyrate; and b) a composition formulated for oraladministration, said composition comprising from about 100 mg to about1000 mg of R-dihydro-lipoic acid to a person or animal in need thereof;wherein said inflammatory bowel disorder is ulcerative colitis.
 2. Themethod according to claim 1, wherein said oral composition isadministered once or twice daily every day, or once or twice daily everyother day.
 3. The method according to claim 1, wherein said enema orsuppository composition is administered once daily, once every two days,once every three days, once every four days, once every five days, onceevery six days, or once every seven days or more.
 4. The methodaccording to claim 1, wherein said oral composition is administereddaily and said enema or suppository composition is administered oncedaily, once every two days, once every three days, once every four days,once every five days, once every six days, or once every seven days ormore.
 5. A kit comprising in one or more containers: i) from about 500mg to about 5000 mg of 5-ASA; and/or ii) from about 1 mg to about 10 mgof budesonide; and/or iii) from about 10 mg to about 1000 mg of cromolynsodium; and/or iv) from about 5 millimoles to about 50 millimoles ofsodium butyrate; and/or v) an emulsifying agent; and/or vi) from about100 mg to about 1000 mg of R-dihydro-lipoic acid.
 6. The method of claim1, wherein said composition formulated for rectal administrationcomprises from about 750 mg to about 3000 mg of 5-ASA; or about 2000 mgof 5-ASA.
 7. The method according to claim 1, wherein said compositionformulated for rectal administration comprises about 15 millimoles ofsodium butyrate.
 8. The method according to claim 1, wherein saidcomposition formulated for rectal administration comprises from about2.5 mg to about 7.5 mg of budesonide; or about 5 mg of budesonide. 9.The method according to claim 1, wherein said composition formulated forrectal administration about 100 mg of cromolyn sodium.
 10. The methodaccording to claim 1, wherein said composition formulated for oraladministration comprises about 250 mg to about 750 mg ofR-dihydro-lipoic acid; or about 300 mg of R-dihydro-lipoic acid.
 11. Anenema formulation comprising: from about 500 mg to about 5000 mg of5-ASA; from about 1 mg to about 10 mg of budesonide; from about 10 mg toabout 1000 mg of cromolyn sodium; and from about 5 millimoles to about50 millimoles of sodium butyrate.
 12. The enema formulation according toclaim 11, further comprising from about 100 mg to about 1000 mg ofR-dihydro-lipoic acid.
 13. A method for the treatment of an inflammatorybowel disorder in a person or animal, said method comprisingadministering once daily an effective amount of: a) a compositionformulated for rectal administration as an enema or suppository in aperson or animal, said composition comprising from about 500 mg to about5000 mg of 5-ASA, from about 1 mg to about 10 mg of budesonide, fromabout 10 mg to about 1000 mg of cromolyn sodium and from about 5millimoles to about 50 millimoles of sodium butyrate; and b) acomposition formulated for oral administration, said compositioncomprising from about 100 mg to about 1000 mg of R-dihydro-lipoic acidto a person or animal in need thereof; wherein said inflammatory boweldisorder is ulcerative colitis.